Kexin-Derived Vaccines to Prevent or Treat Fungal Infections

ABSTRACT

A vaccine is disclosed that promotes CD4+ T cell-independent host defense mechanisms to defend against infection by fungi such as  Pneumocystis  spp. The vaccine may be used to prevent or to treat fungal infections. The novel vaccine can provide protective immunity, even for immunocompromised individuals such as HIV patients having reduced levels of CD4+ T cells.

The benefit of the Jan. 12, 2010 filing date of U.S. provisional patentapplication Ser. No. 61/294,252 is claimed under 35 U.S.C. §119(e) inthe United States, and is claimed under applicable treaties andconventions in all countries.

This invention was made with government support under grant P01-HL076100awarded by the National Institutes of Health. The United StatesGovernment has certain rights in this invention.

TECHNICAL FIELD

This invention pertains to certain proteins derived from kexin, nucleicacids encoding those proteins, and the use of the proteins or nucleicacids as vaccines, for example as vaccines against Pneumocystis jerovicior other Pneumocystis spp.

BACKGROUND ART Epidemiology of Pneumocystis Infection

Despite advances in highly active anti-retroviral therapy (HAART),opportunistic pulmonary infection with Pneumocystis (PC) remains themost common opportunistic infection for HIV patients. Indeed,Pneumocystis pneumonia (PCP) is the index infection for 25-40 percent ofAIDS cases. In patients with established AIDS, prophylactic regimenshave decreased the overall incidence of PCP, but in most patients thismeans that PCP is delayed rather than eliminated. For example, inpatients with CD4 counts below 200/μl who are on recommendedprophylactic regimens, there is still approximately an 18% risk ofactive PCP infection over a 36 month period. The widespread use of PCPprophylaxis also means that more than 80% of PCP cases in the U.S. arenow “breakthrough” cases. Moreover, one study of high-risk childrenfound that the incidence of PCP had not declined despite efforts toidentify HIV-infected infants and to initiate PCP prophylaxis for them.

There is a strong correlation between a higher CD4+ T cell count and alower risk of PCP. Where HAART is successful, as shown by an increase ina patient's CD4+ T-cell count above 200/μl, available data suggest thatPCP prophylaxis can be safely discontinued. Unfortunately, not all AIDSpatients respond to HAART, and drug resistance is emerging. PCP is stilla serious clinical problem in the third decade of the HIV epidemic.There is an unfilled need for improved methods for PCP prevention andtreatment.

In AIDS the depletion or dysfunction of CD4+ lymphocytes not onlyhinders the patient's immune response to infection, it also reduces oreliminates the ability to safely and effectively vaccinate a patientagainst PCP. Pneumocystis is a genus of fungi that is found in therespiratory tracts of many mammals and humans Pneumocystis infection iseasily defended by a healthy immune system. The symptoms of PCPinfection include pneumonia, fever, and respiratory symptoms such as drycough, chest pain and dyspnea. Currently, antibiotics are the preferredmethod of treatment, along with corticosteroids in some severe cases.The most popular antibiotic, and the accepted benchmark for efficacy isTrimethoprim-sulfamethoxazole (TMP-SMX). Alternative antibiotics arealso available due to the severe allergic reactions that some peoplehave to TMP-SMX. Studies have shown that individuals who are on highlyactive antiretroviral therapies (HAART), and who have CD4+ T-cell countsabove a threshold of about 200 cells/mm³ have a sufficient immuneresponse to defend against PCP infection without antibiotics.Prophylaxis is recommended for HIV-positive individuals once their CD4+T cell count falls below 200 cells/mm³, and is also recommended forother severely immunocompromised patients such as transplant patients orleukemia patients. Drug prophylaxis reduces the incidence of PCP andlengthens the disease free intervals between episodes. However, the mosteffective prophylactic treatment, TMP-SMX, has a high rate of adverseeffects. Second-line drugs may be used, but they typically have seriousside effects and generally are less effective.

PCP infection will occur in approximately 15%-28% of individuals withAIDS in a given year. Within the population of HIV/AIDS patients withPCP, the mortality rate is between 10%-20%. An estimated 1 millionpeople worldwide suffer from PCP, while another 5 million people aretreated prophylactically to prevent the disease. Definitive diagnosis ofPneumocystis pneumonia is relatively complex, requiring microscopy oftissues or fluids. As PCP prophylaxis and HAART become more widespread,the incidence of PCP has declined in populations where infection can beproperly diagnosed and treatment can be administered. Studies suggestthat the low prevalence figures reported from developing countries maysimply reflect the lack of adequate infrastructure to properly diagnosePCP.

Organ transplant recipients are also at risk for PCP infection.Transplant recipients take regimens of anti-rejection drugs thatfunction by suppressing the immune system. The overall incidence of PCPin solid organ transplant recipients not taking PCP prophylaxis is about5%, with the highest incidence following liver, heart, and lungtransplants. The most common prophylaxis currently used for organtransplant patients is TMP-SMX, or aerosolized pentamidine if TMP-SMX isnot tolerated by the patient.

Most currently available antibiotic treatments have mild to severe sideeffects, leaving an unfilled need for alternative treatments.Additionally, antibiotic-resistant Pneumocystis are emerging, in partbecause some patients cease treatment due to allergic reaction or otheradverse effects.

Host Defense and Pneumocystis Infection

The inability to reliably culture Pneumocystis organisms in vitro haslimited experimental work with the pathogen to clinical specimens andanimal models of infection. Human Pneumocystis infection is associatedmore with defects in cell-mediated immunity than with neutrophildysfunction. Pneumocystis infections are a particular clinical problemin AIDS patients, whose progressive loss of CD4+ helper T lymphocytesresults in profound suppression of cell-mediated immunity. The risk ofan HIV-infected adult acquiring PCP shows an inverse, and almost linearcorrelation with the number of circulating CD4+ lymphocytes. A similarrelationship has also been seen for in pediatric PCP infection, althoughthe relative CD4+ count may be higher in children. The importance ofCD4+ T lymphocytes in host defense against PCP is further supported bywork with animal models. For example, experimental work from ourlaboratory shows that normal mice inoculated with P. murina are able toresolve the infection without treatment, while mice that have beenspecifically and selectively depleted of CD4+ T lymphocytes with ananti-CD4 monoclonal antibody develop progressive PCP. Whenadministration of the antibody cease and CD4+ lymphocytes are restored,P. murina organisms are cleared from lung tissue and the PCP infectionresolves.

CD4+ T-Cell Factors in Pneumocystis Infection

Among the mechanisms used by CD4+ lymphocytes to mediate host defenseagainst Pneumocystis is the secretion of cytokines such as interferon(IFN). Lymphocytes exposed to PC organisms or to the major surfaceglycoprotein of PC in vitro will secrete IFN. However, lymphocytes fromAIDS patients have a reduced capacity to produce IFN after antigenic ormitogenic stimulation. Although IFN is not directly lethal toPneumocystis, it can activate macrophages in vitro to kill the organism.However, evidence for an in vivo role for IFN in host defense isconflicting. In vivo neutralization of IFN with an antibody has beenreported not to alter clearance of P. murina in reconstituted SCID mice.Also, SCID mice that had been reconstituted with splenocytes from micewith a homozygous deletion of the IFN gene were nevertheless able toreduce levels of P. murina infection.

It has been postulated that a potential target cell for exogenous IFN isthe alveolar macrophage cell, because aerosolized IFN will augmentexpression of these cells. It has been demonstrated that depletion ofalveolar macrophages leads to delayed clearance of P. carinii from therat lung.

Possible mechanisms for IFN bolstering of host defense includeup-regulation of TNF production, increased generation of superoxide, andincreased release of reactive nitrogen intermediates.

Overexpression of interferon by gene delivery results in augmentedclearance of P. murina, which depends in part on enhanced recruitment ofCXCR3+ CD8+ T-cells. Although IFN is clearly therapeutic, endogenous IFNis not required; for example, IFN-gamma knockout (KO) mice can clear P.murina infection.

CD40L and T- and B-Cell Immune Responses and Host Defense Against PC

CD40L is another factor that is expressed on CD4+ T cells, and that iscritical for host defense against PCP. CD40L (also known as CD154) is a33 kDa, type II membrane protein. It is a member of the tumor necrosisfactor (TNF) gene family, and it is a ligand for CD40 on antigenpresenting cells (APC) such as dendritic cells (DCs) and B cells. It hasbeen recently shown that CD40L expression in CD4+ T cells is criticalfor T cell “help,” and permits direct interactions between APCs and CD8+cytotoxic T cells. Moreover, as CD40 is also expressed on B cells,up-regulation of CD40L on CD4+ T cells also is a critical component of Thelper function to induce B cell proliferation.

CD40L:CD40 interactions appear critical for effective host defenseagainst PC. Patients with missense or nonsense mutations in CD40L oftenhave hyper-IgM syndrome. Hyper-IgM syndrome results from a lack ofB-cell differentiation. Patients with hyper-IgM syndrome are ofteninfected with PC. Antibody blockage of CD40L:CD40 interactions preventssplenocyte-reconstituted scid mice from clearing PCP infection. Indeed,4-6 week old CD40L knockout mice from a respected laboratory have beeninadvertently shipped infected with PC. Soluble CD40L has been reportedto have a beneficial effect against PCP in a steroid-inducedimmunosuppressed rat model.

DCs genetically engineered to express CD40L have been reported topresent antigens (from Pseudomonas aeruginosa) to B-cells both in vitroand in vivo in a CD4-independent manner. The resulting antibodiesconferred protection against in vivo challenge with the bacteria.

Our laboratory has previously reported the use of kexin, which is a PCantigen, in a DNA vaccine with or without CD40L. See M. Zheng et al.,“CD4+ T cell-independent DNA vaccination against opportunisticinfections,” J. Clin. Invest., vol. 115, pp. 3536-3544 (2005). Despitethe promise of Kex1 DNA vaccination, there remains an unfilled need forimprovements to the earlier vaccine. Vaccination with the Kex1 DNAresulted in only a 2-3 log improvement in protection as compared tocontrols; mice challenged after Kex1 vaccination still have detectableinfection histologically at 28 days post-PC challenge.

Rationale for a Pneumocystis Vaccine

The pathogenesis of HIV infection involves profound immunosuppression,which leads to greatly increased susceptibility to infections. Mostopportunistic infections in HIV patients involve the respiratory tract.Pneumonia caused by the fungal pathogen Pneumocystis jirovecii remainsthe most common AIDS-defining opportunistic infection. Antimicrobialtherapies are available, but emerging antimicrobial resistance is makingtreatments less effective. Furthermore, high drug costs can precludeantimicrobial therapy in many third world countries have high rates ofHIV infection. Even in developed countries, 20-30% of eligible patientsdo not receive prophylaxis, either because of noncompliance or becauseof the cost of the medications Also, Pneumocystis colonization is nolonger confined to the HIV-infected population. Pneumocystis spp. areincredibly successful pathogens, being found in all areas of the worldand in numerous animal species. PCP infection carries a high mortalityrate. There remains a pressing, unfilled need for new vaccines andvaccination approaches to prevent or treat HIV-associated pulmonaryinfections.

Molecular techniques have recently shown that Pneumocystis colonizationof the respiratory tract is common in many non-HIV-associated pulmonarydiseases, such as emphysema, where PCP can lead to a systemicinflammatory response and accelerated progression of obstructive airwaydisease. Thus, a vaccine against Pneumocystis can prevent not just thedevelopment of pneumonia, but may also limit co-morbidities of HIVinfection, emphysema, and other diseases.

Potential candidates to receive a Pneumocystis vaccine would includeindividuals who are currently candidates for PCP prophylaxis, such asHIV-infected persons with a CD4 count below 200; and patients receivingimmunosuppressive drugs including high-dose corticosteroids, andreceiving anti-inflammatory agents such as anti-TNF andanti-B-lymphocyte agents. Such patients would include transplantrecipients, cancer patients (including leukemia and lymphoma patients),and patients with inflammatory and autoimmune diseases such asrheumatoid arthritis, lupus, or Crohn's disease.

Despite the long-standing need for a vaccine against Pneumocystis orother fungal pathogens, to our knowledge no fungal vaccine has yetreached Phase III clinical trials.

DISCLOSURE OF THE INVENTION

We have discovered a vaccine that promotes CD4+ T cell-independent(CD4IND) host defense mechanisms to defend against infection byPneumocystis and other fungi. The vaccine may be used to prevent or totreat fungal infections, including but not limited to Pneumocystis spp.The novel vaccine can provide protective immunity, even forimmunocompromised individuals with reduced levels of CD4+ T cells.

We used an animal model that mimics HIV-induced CD4+ T cell deficiency:a CD4-depleted mouse treated with GK1.5, which is a monoclonal antibodythat causes 97% or greater depletion of CD4+ T cells in spleen, blood,thymus, and lung. We have shown that using CD40L as an adjuvant allowsthe generation of protective humoral immune responses, even inCD4-deficient patients. We identified immunodominant antigens, includingKex1, a subtilisin-like protease. Mice that were immunized with Kex1cDNA via a DNA-adenovirus vaccine showed significant protection againstPC challenge. Surprisingly, when the vaccine was administered with themolecular adjuvant CD40L, even mice with CD4+ T-cells could develop asubstantial immune response. By contrast, without the CD40L adjuvant,there was a poor response in CD4+ T-cell deficient mice.

We have improved the Kex1 DNA vaccine by defining and isolating asmaller antigen, which we have named “mini-kexin.” This antigen willconfer protective immunity, especially (but not only) when administeredwith a CD40L adjuvant. The mini kexin motif represents a highlyconserved segment across Pneumocystis spp., and homologs are expressedin other fungi. It thus may also provide some protection againstinfection by other Pneumocystis spp. or other fungi, although we havenot yet specifically tested efficacy against other fungal species. Codonoptimization is preferred to enhance the expression of mini kexin DNA ineukaryotic cells; preliminary studies suggest that vaccine efficacy isimproved with the codon-optimized version.

We have also constructed recombinant adenoviruses whose DNA encodesmini-kexin. In preliminary studies these adenovirus-based vectors haveshown greater efficacy and have provoked greater mucosal IgA and IgG2aresponses in the lung, either as compared to DNA alone, or as comparedto systemic boosting with adenovirus. In SIV-infected macaques we haveexamined both anti-Kex1 titers and the rate of PC lung infection, thelatter as determined by nested PCR in BAL fluid. In a cohort of 12macaques, 75% (9 of 12) became PCP positive within 3 months of SIVinfection. The three animals that remained PCP-negative (as determinedby PCR in BAL fluid) had mean serum anti-Kex1 Ab levels that were atleast 10-fold greater than those in PCP-positive monkeys.

CD40 ligand (CD40L) is expressed on activated CD4+ T cells. CD40L and iscritical for host defense against PC as well as bacterial pneumonia. Wehave previously shown that bone-marrow derived dendritic cells (DCs) canbe genetically engineered to express CD40L (using an E1-deletedadenovirus, AdCD40L), resulting in significant DC activation andmaturation. The activated, mature DCs, when pulsed with PC or bacterialantigens, and then injected into mice, produce protective,antigen-specific IgG independently of CD4+ T cells. This strategy wasprotective against PC both in primary-vaccinated, CD4+ T cell-deficientmice, and also in CD4+ T cell-deficient mice receiving adoptive transferof immune serum or CD19+ cells from vaccinated mice. These resultsdemonstrated the critical role of B cells in protecting against PC afterDC vaccination. We have also observed that dendritic cell IL-23 (but notIL-12) is required for functional recall antibody responses to PCantigen challenge. DC-based vaccination can suffer from problems such asscalability, and the cost of producing patient-specific DC's. To try toavoid these problems we developed a prime boost vaccination platformthat greatly enhanced protection against PC in CD4-depleted mice, usingthe immunodominant antigen from PC, Kexin, and CD40L as a molecularadjuvant. Both components were required for vaccine efficacy inCD4-deficient hosts; however, the vaccine only resulted in a 3-logreduction of organism burden and thus did not afford completeprotection.

We then undertook two approaches to improve the effectiveness of thekexin DNA vaccine. One was to use antibody response to map regions ofkexin that were particularly immunogenic. These results showed that over70% of the antibody response was directed against a region of PC kexinthat is highly conserved region across mouse, rat, monkey, and humanPneumocystis spp. We call this 100 amino acid region “mini Kexin.” Thesecond strategy was to perform mucosal boosting rather than systemicboosting. In preliminary studies, we found that mucosal boosting with arecombinant adenovirus encoding mini Kexin afforded significant greaterprotection against PC challenge as compared to systemic boosting.

We discovered that activation of CD40 signaling in vivo, in conjunctionwith vaccination with miniKexin, can produce antigen-specific immuneresponses, even in the absence of CD4+ T-cells. We have furtherdiscovered that mucosal boosting can provide effective vaccinationagainst PCP, even in the absence of CD4+ T-cells.

In one aspect, the vaccine is used for therapeutic purposes in early HIVinfection, when CD4 numbers remain intact, or it is used in otherwiseimmunocompetent hosts who are at risk for infection (e.g., patients withCOPD, cystic fibrosis, or interstitial lung disease). In another aspect,vaccination is administered to individuals with advanced HIV infection,or to other immunosuppressed patients having low numbers of circulatingCD4+ T-lymphocytes, to provide protective immunity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts serum levels of anti-PC antibodies following vaccinationwith different constructs.

FIG. 2 depicts anti-PC IgG2a titers both with and without CD40L.

FIG. 3 depicts PC burden in mice receiving various vaccines, both withand without CD40L.

FIG. 4 depicts PC copy number in the lung 28 days after PC challenge inmice receiving various vaccines, both with and without CD40L.

FIG. 5 depicts IgG titers in mice receiving various vaccines, both withand without CD40L.

FIG. 6 depicts IgA titers in mice receiving various vaccines, both withand without CD40L.

FIG. 7 depicts the induction of IL-12p40, IL-12p70, and IL-23 in micetransduced with AdCD40L, versus controls.

FIGS. 8A, 8B, and 8C depict, respectively, anti-PC IgG1 titers, anti-PCIgG2 titers, and percent killing of PC organisms by anti-PC serum inresponse to vaccination with various DCs.

FIG. 9 depicts the results of PC challenge in prime-boost vaccinatedmice that were artificially immunosuppressed.

MODES FOR PRACTICING THE INVENTION

Preferably, the vaccine comprises a live recombinant delivery system,such as a bacterium or virus expressing mycobacteria genes, or animmunogenic delivery system such as a DNA vaccine, e.g. a plasmid,expressing one or more genes or gene fragments for mini-Kexin.Alternatively, the vaccine may comprise a protein vaccine, that is, themini-Kexin polypeptide itself or a portion thereof, in a delivery systemincluding a carrier or an adjuvant.

In one embodiment, one aspect of the invention is an isolated nucleicacid, preferably DNA, wherein said isolated nucleic acid:

-   -   (a) comprises a sequence that encodes mini-Kexin or a portion        thereof, or comprises a sequence complementary thereto; but does        not encode the entire Kexin protein; or    -   (b) has a length of at least 10 nucleotides, and preferably at        least 20 nucleotides, and hybridizes readily under stringent        hybridization conditions with a nucleotide sequence as disclosed        herein, or with a nucleotide sequence selected from a sequence        described in part (a) above.

Another embodiment comprises such a nucleic acid fragment inserted intoa vector. The vector-based vaccine causes in vivo expression ofmini-Kexin or a portion thereof by a human or other mammal to whom thevaccine has been administered, the amount of expressed antigen beingeffective to confer substantially increased resistance to a pathogenicfungus such as Pneumocystis.

Another embodiment of a vaccine for immunizing a human or other mammalagainst a pathogenic fungus such as Pneumocystis comprises as theeffective component a non-pathogenic microorganism, wherein at least onecopy of a DNA fragment comprising a DNA sequence encoding mini-Kexin ora portion thereof has been incorporated into the microorganism (e.g.,placed on a plasmid or in the genome) in a manner allowing themicroorganism to express, and optionally to secrete mini-Kexin or aportion thereof.

Another embodiment comprises a replicable expression vector thatcomprises a nucleic acid fragment according to the invention, and atransformed cell harboring at least one such vector.

Another embodiment comprises a method for immunizing a mammal, includinga human being, against a pathogenic fungus such as Pneumocystis,comprising administering to the mammal an effective amount of a vaccinea nucleic acid, a polypeptide, a vector, or a cell as described.

A further embodiment comprises a pharmaceutical composition thatcomprises an immunologically reactive amount of at least one memberselected from the group consisting of:

-   -   (a) the mini-Kexin polypeptide, or an immunogenic portion        thereof;    -   (b) a polypeptide whose amino acid sequence has an identity of        at least 70%, 75%, 80%, 85%, 90%, or 95% to any one of said        polypeptides in (a); and is immunogenic;    -   (c) a fusion polypeptide comprising at least one polypeptide        according to (a) or (b) and at least one fusion partner;    -   (d) a nucleic acid that encodes a polypeptide according to        (a), (b) or (c);    -   (e) a nucleic acid whose sequence is complementary to the        sequence of a nucleic acid according to (d);    -   (f) a nucleic acid sequence having a length of at least 10        nucleotides, or at least 20 nucleotides, that hybridizes under        stringent conditions with a nucleic acid according to (d) or        (e); and    -   (g) a non-pathogenic micro-organism that has incorporated        therein (e.g. placed in a plasmid or chromosome) a nucleic acid        sequence according to (d), (e), or (f) in a manner to permit        expression of the encoded polypeptide.

A further embodiment comprises a method for stimulating an immunogenicresponse in an human or other mammal by administering to the human orother mammal an effective amount of at least one member selected fromthe group consisting of:

-   -   (a) the mini-Kexin polypeptide, or an immunogenic portion        thereof;    -   (b) a polypeptide whose amino acid sequence has an identity of        at least 70%, 75%, 80%, 85%, 90%, or 95% to any one of said        polypeptides in (a); and is immunogenic;    -   (c) a fusion polypeptide comprising at least one polypeptide        according to (a) or (b) and at least one fusion partner;    -   (d) a nucleic acid that encodes a polypeptide according to        (a), (b) or (c);    -   (e) a nucleic acid whose sequence is complementary to the        sequence of a nucleic acid according to (d);    -   (f) a nucleic acid sequence having a length of at least 10        nucleotides, or at least 20 nucleotides, that hybridizes under        stringent conditions with a nucleic acid according to (d) or        (e); and    -   (g) a non-pathogenic micro-organism that has incorporated        therein (e.g. placed in a plasmid or chromosome) a nucleic acid        sequence according to (d), (e), or (f) in a manner to permit        expression of the encoded polypeptide.

Definitions. Unless context clearly indicates otherwise, the followingdefinitions should be understood to apply throughout the specificationand claims. Other terms, those for which specific definitions are notgiven, should be interpreted as they would be understood, in context, bya person of skill in the art:

The word “polypeptide” or “protein” or “peptide” should have its usualmeaning: an amino acid chain of any length, including a full-lengthprotein, oligopeptide, short peptide, or fragment thereof, wherein theamino acid residues are linked by covalent peptide bonds. As used in thepresent specification and claims, unless context clearly indicatesotherwise, no distinction is intended between the terms “polypeptide,”“peptide,” and “protein,” which should be considered synonymous.

The polypeptide may be chemically modified by being glycosylated,phosphorylated, I ipidated, by incorporating one or more prostheticgroups or functional group, or by containing additional amino acids suchas e.g. a his-tag or a signal sequence.

Each polypeptide may thus be characterized by specific amino acids andbe encoded by specific nucleic acid sequences. It will be understoodthat such sequences include analogues and variants produced byrecombinant or synthetic methods wherein such polypeptide sequences havebeen modified by substitution, insertion, addition or deletion of one ormore amino acid residues in the recombinant polypeptide and are stillimmunogenic. Substitutions are preferably conservative.

A “substantially pure polypeptide fragment” means a polypeptidepreparation that contains at most 5% by weight of other polypeptidematerial (lower percentages of other polypeptide material are preferred,e.g. at most 4%, at most 3%, at most 2%, at most 1%, and at most ½%). Itis preferred that the substantially pure polypeptide is at least 96%pure, i.e. that the specified polypeptide constitutes at least 96% byweight of total polypeptide material present in the preparation, andhigher percentages are preferred, such as at least 97%, at least 98%, atleast 99%, at least 99.25%, at least 99.5%, and at least 99.75%. It isespecially preferred that the polypeptide fragment is in “essentiallypure form”, i.e. that the polypeptide fragment is essentially free ofany other antigen with which it is natively associated, i.e. essentiallyfree of any other antigen from the same fungus. This can be accomplishedby preparing the polypeptide fragment by means of recombinant methods ina non-fungal host cell, or by synthesizing the polypeptide fragment bythe well-known methods of solid or liquid phase peptide synthesis, e.g.by the method described by Merrifield or variations thereof.

The term “nucleic acid fragment” (or “nucleic acid sequence”) means anynucleic acid molecule including DNA, RNA, LNA (locked nucleic acids),PNA, RNA, dsRNA and RNA-DNA-hybrids. A preferred nucleic acid for use inthis invention is DNA. Also included are nucleic acid moleculescomprising non-naturally occurring nucleosides. The term includesnucleic acid molecules of any length, e.g. from 10 to 10,000nucleotides, depending on the use and context. The nucleic acid moleculeis optionally inserted into a vector.

The term “stringent” when used in conjunction with hybridizationconditions has the meaning generally understood in the art, i.e. thehybridization is performed at a temperature not more than 15-20° C.under the melting point T_(m), cf. Sambrook et al, 1989, pages11.45-11.49. Preferably, the conditions are “highly stringent”, i.e.5-10° C. under the melting point T_(m).

The term “sequence identity” indicates a quantitative measure of thedegree of homology between two amino acid sequences of equal length orbetween two nucleotide sequences of equal length. The two sequences tobe compared are aligned to the best possible fit, allowing for theinsertion of gaps or alternatively, for truncation at one or both ends.The sequence identity can be calculated as (N_(ref)−N_(dif))100/N_(ref),wherein N_(dif) is the total number of non-identical residues in the twosequences when aligned, and wherein N _(ref) is the number of residuesin one of the sequences. Hence, the DNA sequence AGTCAGTC has a sequenceidentity of 75% with the sequence AATCAATC (N_(dif)=2 and N_(ref)=8). Agap is counted as non-identity of the specific residue(s), i.e. the DNAsequence AGTGTC has a sequence identity of 75% with the DNA sequenceAGTCAGTC (N_(dif)=2 and N_(ref)=8). Sequence identity can alternativelybe calculated by available software, such as BLAST, e.g. the BLASTPprogram (Pearson, 1988, or available through ncbi.nlm.nih.gov).Alignment may also be performed with the sequence alignment methodClustalW with default parameters as described by Thompson J., et al1994, available at http://www2.ebi.ac.uk/clustalw/.

A preferred minimum percentage of sequence identity is at least 80%,such as at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, and at least 99.5%.

“Variants.” A common feature of the polypeptides of the invention istheir capability to induce an immunological response. It is understoodthat a variant of min i-Kexin produced by substitution, insertion,addition or deletion may also be immunogenic as determined by any of theassays described herein.

An “immune individual” is a human or other mammal who has cleared orcontrolled an infection with a virulent fungus such as Pneumocystis, orwho has received a vaccination in accordance with this invention.

The “immune response” of an individual may be monitored by any one ofseveral methods known in the art, including for example one or more ofthe following:

A cellular response may be determined in vitro by induction of therelease of a relevant cytokine such as IFN-γ from, or the induction ofproliferation in lymphocytes withdrawn from a human or other mammalcurrently or previously infected with virulent fungus or directly orindirectly immunized with polypeptide. The induction may be performed bythe addition of the polypeptide or an immunogenic portion of thepolypeptide to a suspension comprising from 2×10⁵ cells to 4×10⁵ cellsper well. The cells are isolated from blood, the spleen, the liver, orthe lung, and the addition of the polypeptide or the immunogenic portionresults in a concentration of not more than 20 μg per ml in suspension,with the stimulation being performed over a period from two to fivedays. To monitor cell proliferation the cells are pulsed withradioactively-labeled thymidine; after 16-22 hours of incubation liquidscintillation counting is used to assess proliferation. A positiveresponse is considered to be one that exceeds background by at least twostandard deviations. The release of IFN-γ can be determined, e.g., bythe ELISA method, or other methods known in the art. Other cytokinesbesides IFN-γ may be used to assess immunological response to thepolypeptide, such as IL-12, TNF-α, IL-4, IL-5, IL-10, IL-6, or TGF-β. Asensitive method for detecting an immune response is the ELISpot methodfor determining the frequency of IFN-γ producing cells. In an ELlspotplate (MAHA, Millipore) that is pre-coated with anti-murine IFN-γantibodies (PharMingen), graded numbers of cells isolated from blood,spleen, or lung (typically from 1 to 4×10⁵ cells/well) are incubated for24-32 hrs in the presence of the polypeptide or an immunogenic portiontherefor, at a concentration not more than about 20 μg per ml. Theplates are subsequently incubated with biotinylated anti-IFN-γantibodies followed by a streptavidin-alkaline phosphatase incubation.The cells producing IFN-γ are identified by adding BCIP/NBT (Sigma), andthe relevant substrates develop spots. These spots can be enumeratedwith a dissection microscope. It is also possible to determine thepresence of mRNA that encodes the relevant cytokine by PCR. Usually oneor more cytokines will be measured using, for example, PCR, ELISPOT, orELISA. It will be appreciated by a person skilled in the art that theimmunological activity of a particular polypeptide can be evaluated byobserving whether there is a significant increase or decrease in theamounts of these cytokines.

A cellular response may also be determined in vitro with T cell linesderived from an immune individual, or a Pneumocystis-infected person,where the T cell lines have been driven with either live fungus,extracts from the fungus, or culture filtrate for 10 to 20 days, withthe addition of IL-2. The induction is performed by adding not more than20 μg polypeptide per ml suspension to the T cell lines, from 1×10⁵cells to 3×10⁵ cells per well, with incubation from two to six days. Theinduction of IFN-γ or the release of another relevant cytokine isdetected by ELISA. The stimulation of T cells can also be monitored bydetecting cell proliferation using radioactively labeled thymidine asdescribed above. For both assays a positive response is considered to beone that is at least two standard deviations above background.

A humoral response may be determined in vitro by a specific antibodyresponse from an immune or infected individual. The presence ofantibodies may be determined through methods known in the art, e.g., byELISA or Western blot. The serum is preferably diluted in PBS from 1:10to 1:100 and added to the adsorbed polypeptide, with incubation from 1to 12 hours. By the use of labeled secondary antibodies the presence ofspecific antibodies can be determined by measuring the OD, e.g. byELISA, where a positive response is considered to be one that is atleast two standard deviations above background, or alternatively by avisible response in a Western blot.

Protein Vaccine. Another aspect of the invention pertains to a vaccinecomposition comprising the mini-Kexin polypeptide, or an immunogenicportion thereof, or a fusion polypeptide thereof. It is preferred thatthe vaccine additionally comprise an immunologically andpharmaceutically acceptable carrier, vehicle or adjuvant.

Suitable carriers for polypeptides may be selected from the groupconsisting of a polymer to which the polypeptides are bound by ahydrophobic, non-covalent interaction, such as a polystyrene, or apolymer to which the polypeptides are covalently bound, such as apolysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin,or keyhole limpet haemocyanin. Suitable vehicles may be selected fromthe group consisting of a diluent and a suspending agent. The adjuvantis preferably selected from the group consisting ofdimethyldioctadecylammonium bromide (DDA), Quil A, poly I:C, aluminumhydroxide, Freund's incomplete adjuvant, IFN-γ, IL-2, IL-12,monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), TrehaloseDibehenate, and muramyl dipeptide (MDP).

The preparation of vaccines that contain polypeptides as their activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231 and 4,599,230, andpublished application US2004/0057963, the complete disclosures of all ofwhich are incorporated herein by reference.

Other methods of achieving adjuvant effect for a vaccine include the useof agents such as aluminum hydroxide or aluminum phosphate (alum),synthetic polymers of sugars (Carbopol), aggregation of the polypeptidein the vaccine by heat treatment, aggregation by reactivating withpepsin-treated (Fab) antibodies to albumin, mixture with bacterial cellssuch as C. parvum or endotoxins or other lipopolysaccharide componentsof gram negative bacteria, emulsion in physiologically acceptable oilvehicles such as mannide mono-oleate (Aracel A), or emulsion with 20percent solution of a perfluorocarbon (Fluosol-DA) used as a blocksubstitute. Other possibilities involve the use of immune-modulatingsubstances such as cytokines or synthetic IFN-γ inducers such as polyI:C in combination with an adjuvant.

Another possibility for achieving adjuvant effect is to conjugate thepolypeptide or a portion thereof to an antibody (or antigen bindingantibody fragment) against the Fcγ receptors on monocytes/macrophages.

The vaccines are administered in a manner that is compatible with thedosage formulation, and in an effective, immunogenic amount. Thequantity to be administered depends on the subject to be treated,including, e.g., the capacity of the individual's immune system to mountan immune response, and the degree of protection desired. Suitabledosage ranges are of the order of several hundred micrograms activeingredient per vaccination with a preferred range from about 0.1 μg to1000 μg, such as in the range from about 1 μg to 300 μg, and especiallyin the range from about 10 μg to 50 μg, as may readily be determined byroutine experimentation such as is well known in the art. Suitableregimens for initial administration and booster shots are also variablebut are typified by an initial administration followed by subsequentinoculations or other administrations.

As used in the specification and claims, an “effective amount” or an“effective dosage” of a vaccine is an amount or dosage, that whenadministered to a patient (whether as a single dose or as part of amulti-dose or boosting regimen) provides protective immunity to aclinically significant degree; or alternatively, to a statisticallysignificant degree as compared to control. “Statistical significance”means significance at the P<0.05 level, or such other measure ofstatistical significance as would be used by those of skill in the artof biomedical statistics in the context of immunization.

The manner of application may be varied. Any of the conventional methodsfor administration of a vaccine are applicable. These can include oralapplication on a solid physiologically acceptable base or in aphysiologically acceptable dispersion, parenterally, by inhalation, byinjection or the like. The dosage of the vaccine will depend on theroute of administration and will vary according to the age of the personto be vaccinated and, to a lesser degree, the size of the person to bevaccinated.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations andinhalable aerosols. For suppositories, traditional binders and carriersmay include, for example, polyalkylene glycols or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1-2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like. Thesecompositions may take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders andadvantageously contain 10-95% of active ingredient, preferably 25-70%.

DNA Vaccine. In a preferred embodiment, nucleic acid fragments inaccordance with the invention are used for the in vivo expression ofantigens, i.e. in so-called DNA vaccines as reviewed in Ulmer et al1993, which is incorporated by reference. Hence, the invention alsorelates to a vaccine comprising a nucleic acid fragment according to theinvention, the vaccine causing in vivo expression of antigen by a humanor other mammal, the amount of expressed antigen being effective toconfer substantially increased resistance to infections caused byvirulent fungi, including for example Pneumocystis jerovici or otherPneumocystis spp.

Live Recombinant Vaccines; Plasmids. Another possibility for effectivelyactivating a cellular immune response is to express the antigen in anon-pathogenic microorganism or virus that is then used as a vaccine.Well-known examples of such microorganisms are Mycobacterium bovis BCG,Salmonella, and Pseudomonas, and examples of such viruses are VacciniaVirus and Adenovirus.

Accordingly, another aspect of the present invention is to incorporateone or more copies of a DNA sequence as described into the genome of themicroorganism or virus in a manner allowing the micro-organism toexpress and secrete the polypeptide. The incorporation of more than onecopy of a nucleotide sequence of the invention may enhance the immuneresponse.

Another possibility is to integrate the DNA encoding the polypeptide inan attenuated virus such as the vaccinia virus or Adenovirus (Rolph etal 1997). The genes carried by the recombinant vaccinia virus areexpressed within an infected host cell, and the expressed polypeptide ofinterest can induce an immune response.

Because the target population for this vaccine will often have acompromised immune system, even attenuated live vaccines may beinappropriate vehicles. In such cases, it can be preferred to administerthe DNA sequence in a non-replicating vehicle, such as a plasmid or adisabled virus that is capable of delivering DNA to a host cell, butthat is incapable of replicating in the host.

Example 1

Co-administration of CD40L with mini-Kexin vaccination induces a CD4INDhumoral response and protection against PC in vivo. Four forms ofmini-Kexin DNA are used for vaccination: mini-Kexin-wild type(mKexin-WT); mini-Kexin that has been codon optimized for mammalianexpression (mKexin-CO); miniKexin that has been engineered to besecreted with an IgGk leader sequence (smKexin); and smKexin that hasbeen codon optimized (smKexin-CO). We compared these wild type andcodon-optimized forms of the DNA vaccine. We also compare mucosalboosting with recombinant adenovirus and recombinant modified vacciniaAnkara strain (MVA) vectors. Outcome measures include anti-Kexin andanti-PC isotype-specific antibody responses, as well as anti-Kexinsubclass determinations. Serum is tested in functional assays includingopsonic phagocytosis, and passive transfer protection into scid mice. Wealso examine the efficacy of the vaccine against PC challenge performedat several times after vaccination.

Example 2

Our hypothesized mechanism predicts that endogenous IL-23 is required;and results in durable vaccine responses in both CD4+ T-cell deficientmice and CD40L knockout mice. Specifically we demonstrate the efficacyof CD40L co-transduction in CD40L knockout mice; and the requirement ofIL-12 family members (including IL-12p35, IL-12p40, and IL-23), andcritical activation molecules that are induced by CD40L-modified DCs togenerate effective primary and memory B-cell responses. Preliminarystudies have suggested that IL-23 production is critical to generateB-cell memory against PC antigen.

Example 3

CD4IND pathogen-specific immune responses against Pneumocystis kexin aregenerated in an SIV model of immunodeficiency in macaques. We expectthat the mini-kexin constructs will produce vaccine-induced immuneresponses in SIV-infected, CD4 deficient macaques. Control or SIVinfected macaques will undergo DNA priming, followed by mucosal boosting4 weeks after mock or live SIV infection. Outcome measures will includehumoral responses to the vaccine, and the prevention of Pneumocystiscolonization as determined by PCR of BAL fluid. Preliminary studiessuggest that Pneumocystis colonization occurs in up to 80% of SIVinfected macaques, compared to 0% in non-SIV infected monkeys. We willalso challenge SIV-infected monkeys with live Pneumocystis, anddemonstrate vaccine efficacy in the challenge model.

Example 4

We generated anti-Pneumocystis antibodies in CD4-deficient mice byvaccination with PC-pulsed, CD40L-transduced, bone marrow-deriveddendritic cells. These antibodies stain the surface of PC, and enhanceopsonic phagocytosis and killing of PC in a dectin-1-independent butFc-dependent manner. These antibodies also confer significant protectionagainst PC when passively transferred to scid mice prior to PCchallenge.

Example 5

We identified antigen specificities using both 1-dimensional and2-dimensional electrophoresis, as well as immunoprecipitation followedby 2-D gel electrophoresis. Silver-stained spots on 2-D gels werepicked, enzymatically-digested, and analyzed by tandem MS (AppliedBiosystems). We also performed N-terminal sequencing on proteins. Due toa lack of published data for the entire PC genome, and in light of thesignificant homology of many PC genes to those of Saccharomycescerevisiae and S. pombe, we performed homology searches against PC andSaccharomyces spp. One antigen consistently identified by both MS-MS andN-terminal sequencing was kexin (also called Kex1). Kex1 is a proteasewith high homology to furin. Kexin is presumably involved in processingof pre-pro proteins in yeast. Monoclonal antibodies raised against Kex1show protective efficacy in murine models of PCP.

Example 6

We cloned the full length Kex1 cDNA, and generated DNA vaccines, bothwith and without an additional open reading frame encoding CD40L as aB-cell adjuvant. CD4-deficient mice that were immunized by intramuscularDNA encoding Kex1 and CD40L developed significant anti-Pneumocystisantibody titers, as well as approximately a three log protection againstPC challenge. Moreover these antibodies stained the surface of PCorganisms from mouse and monkey, and enhanced opsonic phagocytosis andkilling of mouse PC in vitro. However, despite the efficacy offull-length Kex1 vaccination, vaccinated mice still had readilydetectable infection 4 and 6 weeks after challenge.

Example 7

To improve upon our original Kex1 vaccine we tried several approaches.The first was to examine if mucosal boosting with recombinant adenoviruswould enhance DNA priming. Although full-length Kex1 could be packaged,the recombinant Ad5-based vectors grew poorly, with titers of 10⁷ or 10⁸per ml. The Kex1 coding sequence is over 3 kB. We explored whether wecould improve both packaging and expression by truncating the antigenand by using codon optimization. Our analysis of Kex1 revealed a 100amino acid segment of Kex1 with over 75% homology among PC organismsobtained from mouse, rat, monkey, and human hosts. (See FIG. 1 frompriority application 61/294,252, not reproduced here but incorporated byreference.)

Example 8

Expressing peptides from this region of Kex1 in recombinant E. coli, wedemonstrated that antibodies recognizing epitopes in this region accountfor a significant amount of the opsonic killing of PC. PC organisms wereopsonized with naive serum (control), or with serum from mice vaccinatedwith full length kexin/CD40L, and then incubated with peritonealmacrophages to assess opsonic killing in vitro. To assess viability ofPC organisms 24 hours later, we measured the integrity of the PCmitochondrial large subunit mRNA by real time PCR. Opsonization of PCwith serum from Kexin/CD40L-vaccinated mice markedly increased PCkilling in vitro. Absorption of the serum against Kexin peptides oragainst miniKexin markedly decreased opsonic phagocytosis, suggestingthat recognition of epitopes in this 100 aa stretch of Kexin isimportant to activity. (See FIG. 2 from priority application 61/294,252,not reproduced here but incorporated by reference.)

Example 9

We next performed passive transfer experiments into scid mice usingcontrol serum, serum from Kexin/CD40L vaccinated mice, and serum fromvaccinated mice that had been pre-adsorbed against recombinant Kexin.The mice were then challenged with PC (2×10⁵ cysts) intratracheally.Mice were sacrificed at day 28, and PC burden in the lung was assessedby real-time PCR. Transfer of 300 μL of serum fromKexin/CD40L-vaccinated mice resulted in significantly reduced PC burdenas compared to control serum. Adsorption of serum against recombinantKexin significantly attenuated the protection of the transferred serum.(See FIG. 3 from priority application 61/294,252, not reproduced herebut incorporated by reference.)

Example 10

We modified the vaccine by constructing vectors encoding the 100 aminoacid conserved region of Kex1 that we identified, a region that we havenamed “mini-Kexin.” We constructed 4 DNA vaccines: (1) wild-type miniKexin without a leader sequence, (2) wild type mini Kexin with an IgGkleader sequence to facilitate secretion, (3) codon-optimized mini Kexinwith no leader sequence, and (4) codon optimized mini Kexin with a IgGkleader sequence. These vectors were called, respectively: (1)pmini-Kexin WT, (2) psec-mini-Kexin-WT, (3) pmini-Kexin CO, and (4)psec-mini-Kexin-CO. To assess the secretion of mini-Kexin we transfected293 cells with these constructs and assayed for Kexin by direct ELISA incell lysates or in cell supernatants 48 hours after transfection. (SeeFIG. 4 from priority application 61/294,252, not reproduced here butincorporated by reference.) The addition of the IgG-kappa leadersequence in the psec-mini Kexin constructs resulted in higher levels ofKexin in cell supernatants. Moreover, codon optimization was associatedwith higher expression. Thus a preferred embodiment uses both a leadersequence and codon optimization.

Example 11

We next examined the efficacy of these constructs in DNA vaccination ofCD4-depleted mice. For these studies, mice were vaccinated withpmini-Kexin WT (K-wt), psec-mini-Kexin-WT (sK-wt), pmini-Kexin CO(K-co), or psec-m in i-Kexin-CO (sK-co). The mice were vaccinated withconstructs either lacking CD40L or with a sequence encoding CD40L clonedinto the second open reading frame of the plasmids, to act as a B-celladjuvant. Mice were vaccinated by two injections of 100 μg DNA, givenintramuscularly three weeks apart. Anti-PC antibodies were measured byELISA 7 days after the second injection of DNA. FIG. 1 shows serumlevels for anti-PC antibodies, measured as end point dilutions (1:64).Mice vaccinated with psec-mini-Kexin-CO (leader sequence, codonoptimized) had the highest levels of anti-PC IgG1. Interestingly, thepresence or absence of CD40L seemed to have little effect on anti-PCIgG1 titers (FIG. 1, * denotes p<0.05 compared to SK-wt, K-wt, and K-co,ANOVA, n=6-8 per group). By contrast, the presence of CD40L wasassociated with significant increases in anti-PC IgG2a titers ascompared to constructs lacking CD40L (FIG. 2, * denotes p<0.05 comparedto the non-CD40L group, ANOVA, n=6-8 per group).

Example 12

To test the protective effect of the antibodies against PC infection, weperformed a PC challenge following the second dose of DNA. The mice weresacrificed 28 days later to assess PC organism burden in lung tissue(FIG. 3). Mice vaccinated with psec-kexin-Co-CD40L had the lowestorganism burden in the lung compared to all other groups (*p<0.01 ANOVA,n=6 per group). Furthermore, the addition of CD40L was associated withlower organism burdens in all vaccine groups compared to mice vaccinatedwithout CD40L. Nevertheless, there were still between 10⁶ and 10⁷ PCorganisms present, even in the psec-kexin-Co-CD40L group.

Example 13

We examined whether mucosal boosting can augment protection against PC.We constructed recombinant Ad5-based vectors encoding all four of themini-Kexin constructs described above. Mice were primed with 2 IMinjections of DNA, followed either by no mucosal boost or by intranasalboosting with 10⁷ PFU of Ad5 encoding the same construct as was used forthe DNA prime vaccination. FIG. 4 depicts PC copy number in the lung 28days after PC challenge in these mice. The mucosal boost with AdCD40Lresulted in nearly a three log improvement in both the sK-co and k-Cogroups (p<0.01 ANOVA, n=6 per group, compared to SK-wt or K-wt withCD40L). Immune response was enhanced with CD40L, both when used in theDNA prime as well as when used in the adenovirus boost. The improvedprotection against PC challenge was associated with higher anti-PC IgGtiters (FIG. 5, n=6 each group, * p<0.05 ANOVA) as well as higheranti-PC IgA titers in BAL (FIG. 6, n=6 each group, * p<0.05 ANOVA).These preliminary data suggest that psecKexin-co and pmini-Kexin-co withCD40L are superior vaccines.

Example 14

Our hypothesis predicts that this strategy: (1) should requireendogenous IL-23, and (2) should result in durable vaccine responsesboth in CD4+ T-cell deficient mice and in CD40L knockout mice. We havedemonstrated that AdCd40L is a potent inducer of IL-12p40, IL-12p70, andIL-23 (FIG. 7). For these experiments, bone marrow-derived dendriticcells were grown from hematopoietic progenitors, and transduced with AdLuc or AdCD40L at a dose of 100 viral particles per cell. Supernatantswere collected 24 hours later and assayed for IL-12p40 or IL-12p70 byLuminex, or for IL-23 by ELISA (n=5 per group, * denotes p<0.05 comparedto AdLuc controls).

Example 15

To determine the role of IL-12 and IL-23 in AdCD40L-transduced, DC-basedvaccine responses, we generated DCs from IL-12p40−/−, IL-12p35−/−, orIL-23p19−/− mice; transduced each DC genotype with AdCD40L; pulsed theDCs with PC antigen; and then administered the DCs intravenously toCD4-depleted mice. Primary antibody responses were measured after 4weeks. To assess recall responses, mice were re-challenged with PCantigen by IP injection, and serum antibody responses were measured 10days later. As shown in FIG. 8A, primary IgG1 responses were similarregardless of the DC expression of IL-12p40, IL-12p35, or IL-23p19. Micevaccinated with IL-23-deficient DCs (either IL-12p40−/− or IL-23p19−/−DCs) had reduced primary IgG2a responses to PC (FIG. 8B). Furthermore,recall response to PC antigen, as a measure of B-cell memory, wassignificantly diminished both in IL-12p40−/− and in IL-23p19 deficientDCs, but not in IL-12p35−/− DCs (FIGS. 8A and 8B, * denotes p<0.05 ascompared to the other groups, ANOVA, n=5-6 per group). These datademonstrated that IL-23 is an important mediator of CD40L-induced B cellexpansion and antigen-specific recall responses. The defect infunctional B-cell memory was also associated with diminished opsonickilling activity of anti-PC serum from CD4-depleted mice vaccinated withDCs from either IL-12p40/− or IL-23p19−/− mice (FIG. 8C, * denotesp<0.05 compared to the other groups, ANOVA, n=5-6 per group).

Example 16

CD4IND, pathogen-specific immune responses against Pneumocystis kexinare produced in an SIV model of immunodeficiency in macaques. We haveassessed spontaneous PC infection in macaques infected with SIV/DeltaB670. CD4 counts below 500 cells/μL have been strongly associated withan increase in PC colonization in the lung, as assessed by nested PCR inBAL fluid. Five of five SIV-infected monkeys developed detectable PCcolonization, as assayed by nested PCR. Interestingly, several monkeyshad an initial increase in anti-Kexin antibody titers, followed by afall in titers prior to the development of a positive PCR response forPC. In a second cohort of animals, preliminary studies suggested thatSIV-infected monkeys with high baseline anti-Kex1 titers were protectedagainst PC infection, as measured by PCR in BAL. We will determine therate of PC infection at necropsy in 25 SIV-infected and 25non-SIV-infected macaques. For these studies we will assess PCcolonization by nested PCR, and real-time PCR on lung tissue and BAL.These data will also be compared to standard histological detection ofPC by GMS staining of lung tissue.

Example 17

Additional studies confirm that CD 40L co-administration results inCD4IND immune responses after IM DNA vaccination, through B-cellresponses. Our preliminary results suggest that codon-optimized,secreted antigen is a better driver of the B cell response as comparedto non-secreted or wild-type forms of Kexin. We have cloned the kexinconstructs and CD40L into pBUDCE4.1 (Invitrogen), which contains twoexpression cassettes (one driven by CMV and one driven by the human EF-1alpha promoter), allowing both genes to be effectively expressed intransduced cells

Example 18

Experimental Groups. Male 6-8 week old BALB/c mice will be CD4-depletedby the administration of 0.3 mg GK1.5 IP by weekly injection, or givenrat IgG as a control. 48 hours later, mice will be randomized to bevaccinated by the IM injection of pmini-Kexin WT, psec-mini-Kexin-WT,pmini-Kexin CO, or psec-mini-Kexin-CO; and in each case, either with orwithout CD40L. One group of mice will be injected with pBudCD40L with noPC antigen as a control. Sample size will be 10 mice per group, whichshould give sufficient statistical resolution to detect a ˜30%difference in anti-PC or Kexin IgG between different routes ofvaccination.

Manipulations: There will be two doses of plasmid injections, threeweeks apart.

Measures and Outcomes: Sera will be assayed for anti-PC and anti-Kexinantibodies, both IgM and IgG, by ELISA at 3, 6, and 9 weeks. Isotypeswill be determined by using appropriate anti-mouse IgG isotypes: IgG1,IgG2a, IgG2b, IgG3 (Pierce, Rockford, Ill.). At 9 weeks, mice will besacrificed and lungs will be lavaged for anti-PC and anti-Kexin IgAlevels. Kexin-specific, IgG-expressing B cells in the spleen andmediastinal lymph nodes will be assayed by Elispot. Serum antibodieswill also be tested for complement-dependent killing, and for opsonicphagocytosis and killing of PC in vitro. Briefly, serial dilutions ofsera will be incubated with PC cysts and cultured in RPM1640+10% FCS(with or without heat inactivation) for 24 hours, followed by assessmentof PC mtLSU rRNA integrity by real-time PCR. To assessmacrophage-dependent killing the assay is performed in the presence of50,000 alveolar macrophages obtained by lung lavage. If we observe highantibody titers and augmentation of PC killing in vitro, we will alsoperform passive transfer experiments in scid mice with 300 μL of serum,followed by pulmonary PC challenge. Scid mice will be sacrificed 28 dayslater to determine if the passive transfer of serum prevents PCinfection.

Expected Results and Interpretations: We expect to observe significantinduction of anti-PC and anti-Kexin IgG in CD4-depleted mice vaccinatedwith pmini-Kexin WT, psec-mini-Kexin-WT, pmini-Kexin CO, orpsec-mini-Kexin-CO—particularly when CD40L is included in the plasmid.We also expect that CD40L will be required for efficient IgA productionin BAL in these mice. Our preliminary data also suggest that IgG levels,and perhaps Kexin-specific IgG- and IgA-producing B-cel Is are enhancedin the secreted Kexin/codon optimized, CD40L groups in the spleen. Weexpect the precursor frequency to be significantly lower in themediastinal lymph nodes as compared to spleen in this stage of thevaccine. We also expect to observe vaccine-induced increases in opsonicactivity and killing of PC in vitro, as well as protection in passivetransfer experiments.

Alternative Approaches: We expect to observe a mixed TH1 and TH2antibody response, since IL-4 has been reported to have synergy withIL-12 p70 and CD40L in activating B-cells to a TH2 response.Alternatively, activated DC's have been shown to induce preferential TH1responses in B-cells. Thus we will observe the roles of specificendogenous cytokines such as IL-12, IL-23, in IL-12p40, IL-12p35, andIL-23 knockout mice. If we observe only a modest anti-PC/Kexin IgGresponse in the IM regimen, we will investigate electroporation afterDNA administration. Electroporation enhances CD8+ T-cell responses. Itseffect on humoral immune response is unclear, but and we are alreadyachieving significant B-cell responses, and therefore seeelectroporation as being less preferred. The titer or half life of theantibody in the scid mice will be confirmed by measuring the titer bothimmediately after transfer and on day 28. Prior studies with DC-basedvaccines suggest that 300 μL of serum should provide sufficient Ab toprotect during the 28 day study period.

Example 19

The effect of mucosal boosting with recombinant adenovirus virus-basedvectors following DNA priming.

Hypothesis: We hypothesize that the co-administration of CD40L withantigen allows for CD4-independent (CD4IND) B-cell responses in vivo,and that mucosal boosting will enhance mucosal antigen-specific B-cellresponses, as well as overall protective immunity.

Rationale: Preliminary studies have demonstrated that CD40Lco-administered with Kexin antigen resulted in CD4IND B-cell responses.To optimize the in vivo response, we postulate that mucosal boostingwith recombinant adenovirus vectors will enhance mucosal IgA and IgGB-cell responses.

Experimental Groups. Male 6-8 week old BALB/c mice will be CD4-depletedby the administration of 0.3 mg GK1.5 IP by weekly injection or givenrat IgG as a control for the CD4-replete group. 48 hours later, micewill be randomized to be vaccinated by the IM injection of pmini-KexinWT, psec-mini-Kexin-WT, pmini-Kexin CO, or psec-mini-Kexin-CO, in eachcase with or without CD40L. One group of mice will be injected withpBudCD40L with no PC antigen as a control. After 6 weeks mice will befurther randomized for mucosal boosting with 10⁷ adenovirus encoding thesame antigen construct as the prime vaccination, with or without anequal dose of AdCD40L. Sample sizes will be 10 mice per group, to givethe statistical resolution to detect a 30% difference in anti-PC orKexin IgG between different routes of vaccination.

Manipulations: Plasmid injections will be repeated every three weeks fortwo doses as otherwise previously described by Ramsay et al. We willadminister 10⁷ of E1-deleted, Ad5-based vectors encoding antigen, withor without AdCD40L by intranasal administration.

Measures and Outcomes: Sera will be assayed for anti-PC and anti-KexinIgM, and IgG isotypes by ELISA at 3, 6, and 9 weeks using appropriateanti-mouse IgG isotypes: IgG1, IgG2a, IgG2b, IgG3 (Pierce, Rockford,Ill.). At 9 weeks, mice will be sacrificed and lungs will be lavaged foranti-PC and anti-Kexin IgA levels. B cells expressing Kexin-specific IgGand IgA in the spleen and mediastinal lymph nodes will be assayed byElispot. Serum and BAL antibodies will also be tested forcomplement-dependent killing as well as opsonic phagocytosis and killingof PC in vitro. In brief, serial dilutions of sera will be incubatedwith PC cysts and cultured in RPM1640+10% FCS (with or without heatinactivation) for 24 hours, followed by assessment of PC mtLSU rRNAintegrity by real-time PCR. To assess macrophage-dependent killing theassay is performed in the presence of 50,000 alveolar macrophagesobtained by lung lavage. If we observe high titers of Ab andaugmentation of PC killing in vitro, we will also perform passivetransfer experiments in scid mice with 300 μL of serum followed bypulmonary PC challenge. Scid mice will be sacrificed 28 days later todetermine if the passive transfer of serum prevents PC infection.

Expected Results and Interpretations: We expect to observe significantenhancement in both BAL anti-PC and anti-Kexin IgG and IgA in animalsthat are mucosally boosted as compared to those in Example 18. Moreoverwe expect to observe increases in Kexin-specific IgG and IgA B-cells inthe mediastinal lymph nodes in Ad-boosted mice. We also expect thatCD40L will be required in both the priming and the boosting regimen toachieve strong mucosal IgG and IgA anti-PC and anti-Kexin antibodyresponses. We also expect to observe vaccine-induced increases inopsonic activity and killing of PC in vitro, as well as protection inpassive transfer experiments.

Alternative Approaches: The dosage of the boost will be optimized,following initial proof of concept. The initial dose of 10⁷ has beenvalidated in preliminary studies, but could be increased, e.g., to 10⁸for both antigen-containing Ad as well as for AdCD40L. The titer andhalf life of the antibody in the scid mice will be assayed by measuringtiter immediately after transfer and on day 28. Prior studies withDC-based vaccines suggest that 300 μL of serum should provide sufficientAb to protect throughout the 28 day study period. The relativeimportance of mucosal Ab and serum Ab for protective immunity can alsobe assayed, e.g., by transferring concentrated BAL to supply 100 μg ofprotein. Controls will consist of BAL that is brought up to 100 μg ofprotein with naive mouse serum.

Example 20

The effect of mucosal boosting with recombinant adenovirus virus-basedvectors after DNA priming in conferring protection against a PCchallenge.

Hypothesis: We hypothesize that the co-administration of CD40L withantigen allows for CD4IND B-cell responses in vivo, and that mucosalboosting will enhance mucosal antigen-specific B-cell responses andconfer protection against PCP.

Rationale: Preliminary studies demonstrated that CD40L co-administeredwith Kexin antigen results in CD4IND B-cell responses and protectionagainst PCP. These studies will confirm these preliminary results.

Experimental Groups. The groups will be similar to those used in Example19, but this study will assess responses to PC challenge. Male 6-8 weekold BALB/c mice will be CD4-depleted by the administration of 0.3 mgGK1.5 IP by weekly injection, or given rat IgG as a control. 48 hourslater, mice will be randomized to be vaccinated by IM injection ofpmini-Kexin WT, psec-mini-Kexin-WT, pmini-Kexin CO, orpsec-mini-Kexin-CO, in each case either with or without CD40L. A controlgroup of mice will be injected with pBudCD40L with no PC antigen. After6 weeks mice will be further randomized, and either given no boosting ormucosal boosting with ˜10⁷ adenovirus, encoding the same amount ofantigen as the prime, again, with or without an equal dose of AdCD40L.At 9 weeks mice will be challenged with 2×10⁵ PC cysts and followed for6 weeks to determine PC lung burden by quantitative real time PCR.Sample Sizes will consist of 10 mice per group to give the power toresolve a 30% difference in PC burdens.

Manipulations: Plasmid injections will be repeated every after weeks fortwo doses as otherwise previously described by Ramsay et al. We willadminister 10⁷ of E1-deleted Ad5 based vectors encoding antigen, with orwithout AdCD40L, by intranasal administration. Mice will be sacrificed 6weeks after PC challenge to assay for serum and BAL anti-PC andanti-Kexin antibodies, PC burden by real time PCR, and GMS staining oflung tissue.

Measures and Outcomes: Sera will be assayed for anti-PC and anti-KexinIgM, and IgG isotypes by ELISA at 3, 6, 9 and 15. Isotypes will bedetermined by using appropriate anti-mouse IgG isotypes: IgG1, IgG2a,IgG2b, IgG3 (Pierce, Rockford, Ill.). At sacrifice one lung will beinflated with 10% neutral buffered formalin and sent for morphologyexamination using H & E and GMS staining. The other lung will be placedin TRIzol™ reagent prior to assaying for PC burden by real time PCR.Serum and BAL antibodies will also be tested for complement-dependentkilling, opsonic phagocytosis, and killing of PC in vitro as otherwisedescribed above.

Expected Results and Interpretations: We expect to observe significantprotection in mice vaccinated and boosted with adenovirus carrying DNAthat encodes kexin antigens. Based on preliminary studies we expect toobserve the greatest protection in the codon-optimized, secreted Kexingroup. We expect to achieve a 6-log level of protection compared toCD40L vaccinated control mice without antigen. During the challengestudies we also will incorporate a scid mouse control group to verifyinfection with the dose of PC used. Both the scid group and the control,CD4-depleted mice typically have over 10⁹ PC copy number in their lungby week 6. Thus in the effective vaccine group we expect to observelevels of 10³ PC copy number or lower. We also expect to observevaccine-induced increases in opsonic activity and killing of PC in vitroas well as protection in passive transfer experiments.

Alternative Approaches: The dosage of the boost will be optimized. Theinitial dose of 10⁷ has been validated in preliminary studies, but couldbe increased, e.g., to 10⁸ for both antigen-containing Ad, as well asfor AdCD40L. We will also determine the duration of protection. We willchoose the two most effective vaccine boost combinations (with andwithout CD40L), and challenge at week 16 or week 26 to determine whetherprotection still exists. We will also assess anti-PC and anti-Kexinrecall responses in the lung and serum as well as B-cell Elipsots toassess functional B cell memory. Here we expect that CD40L will berequired for long term functional B-cell responses. To confirm that thetiter and half life of the antibody suffice to protect the scid mice, wewill measure the titer immediately after transfer, and on day 28. Priorstudies with DC-based vaccines suggest that 300 μL of serum shouldprovide sufficient Ab to protect during the 28 day study period Therelative importance of mucosal Ab and serum Ab to protective immunitycan also be assayed, e.g., we could transfer concentrated BAL containing100 μg of protein. Controls will consist of BAL that is brought up to100 μg of protein with naive mouse serum.

Example 21

Effect of mucosal boosting with recombinant MVA virus-based vectorsafter DNA priming in conferring protection against a PC challenge.

Hypothesis: We hypothesize that the co-administration of CD40L withantigen allows for CD4IND B-cell responses in vivo, and that mucosalboosting with modified vaccinia Ankara strain (MVA)-based vectors willenhance mucosal antigen specific B-cell responses and confer protectionagainst PCP.

Rationale: Our preliminary studies have demonstrated that CD40Lco-administered with Kexin antigen resulted in CD4IND B-cell responsesand protection against PCP. However there could be a concern that anAd5-based vector might itself exacerbate HIV disease in patients withpre-existing Ad5 antibodies. Therefore, although our pre-clinical datasupport the efficacy of Ad5-based vectors in rodents, it is possiblethat an alternative approach could be a useful option for at least somepatients with HIV disease. We chose MVA vectors to explore such analternative, as MVA elicits strong mucosal immune responses.

Experimental Groups. Male 6-8 week old BALB/c mice will be CD4-depletedby the administration of 0.3 mg GK1.5 IP by weekly injection, or givenrat IgG as a control. 48 hours later mice will be randomized to bevaccinated by the IM injection of pmini-Kexin WT, psec-mini-Kexin-WT,pmini-Kexin CO, or psec-mini-Kexin-CO, in each case with or withoutCD40L. A control group of mice will be injected with pBudCD40L with noPC antigen. After 6 weeks mice will be further randomized, and giveneither no boost or mucosal boosting with 10⁷ MVA, encoding the sameantigen construct as the prime, in each case either with or without anequal dose of MVA CD40L. A subgroup of mice will be sacrificed todetermine pre-challenge antibody and B cell responses as outlined inExample 20, and the other mice will be challenged with 2×10⁵ PC cystsand followed for 6 weeks. Sample sizes will consist of 10 mice per groupto give the power to resolve a 30% difference in PC lung burden byquantitative real time PCR.

Manipulations: Plasmid injections will be repeated with a second doseafter three weeks, as otherwise previously described by Ramsay et al. Wewill administer 10⁷ of MVA vectors encoding antigen, either with orwithout MVA CD40L, by intranasal administration. Mice will be sacrificed6 weeks after PC challenge to determine serum and BAL anti-PC andanti-Kexin antibodies, PC burden by real time PCR, and GMS staining oflung tissue.

Measures and Outcomes: Sera will be assayed for anti-PC and anti-KexinIgM, and IgG isotypes by ELISA at 3, 6, 9 and 15 weeks. Isotypes will bedetermined by using appropriate anti-mouse IgG isotypes: IgG1, IgG2a,IgG2b, IgG3 (Pierce, Rockford, Ill.). At sacrifice one lung will beinflated with 10% neutral buffered formalin and sent for morphologyexamination using H & E and GMS staining. The other lung will be placedin TRIzol™ reagent to assay PC burden by real time PCR.

Expected Results and Interpretations: We expect to observe significantprotection in mice vaccinated and boosted with MVA vectors encodingkexin antigens. Based on our preliminary studies we expect the greatestprotection will occur in the codon-optimized, secreted Kexin group. Ourgoal is to achieve a 6-log level of protection as compared to thecontrol mice vaccinated with CD40L without antigen. During the challengestudies we also will incorporate a scid mouse control group to verifyinfection with the dose of PC used.

Alternative Approaches: The dosage of the boost will be optimized. Theinitial dose of 10⁷ has been validated in preliminary studies, but couldbe increased, e.g., to 10⁸ for both antigen and CD40L. If we observesignificant protection, we will next determine the duration ofprotection. We will choose the two most effective vaccine boostcombinations (with and without CD40L) and challenge at week 16 or week26 to determine whether protection still exists. In these studies wewill also assess anti-PC and anti-Kexin recall responses in lung andserum, as well as B-cell Elipsots to assess functional B cell memory.Here we expect that CD40L will be required for long term functionalB-cell responses.

Example 22

Our hypothesis predicts that effective vaccination requires endogenousIL-23, and results in durable vaccine responses in both CD4+ T-celldeficient mice and CD40L knockout mice. We will examine the efficacy ofCD40L co-transduction in CD40L knockout mice; and the requirement ofIL-12 family members (including IL-12p35, IL-12p40, and IL-23), allcritical activation molecules that are induced by CD40L-modified DCs ingenerating effective primary and memory B-cell responses. Ourpreliminary studies have suggested that IL-23 production is critical togenerate B-cell memory against PC antigen.

Example 23

We investigate whether co-administration of CD40L with prime-boostvaccination can induce Ig class switching in CD40L knockout mice, andresult in antigen-specific B-cell responses.

Hypothesis: We hypothesize that CD40L co-transduction with Kexin willresult in class switching of B cells in CD40L KO mice and the generationof anti-Kexin IgG.

Rationale: Patients with mutations in CD40L resulting in Hyper-IgMsyndrome are often infected by PC. There is an unfilled need foreffective vaccines for these individuals.

Experimental Groups. Male PC-free 6-8 week old CD40L knockout or controlmice will be randomized to be vaccinated by the IM injection of pKexinbased on the two most efficacious constructs identified above, followedby randomization for no boost, or boosting with AdKexin, or boostingwith AdKexin/CD40L, or injection with AdCD40L alone as a control. Samplesizes will consist of 10 mice per group to give the resolution to detecta 30% difference in anti-Kexin or anti-PC IgG between different routesof vaccination. The optimal dose and route of the DNA prime and boostvector will be chosen based on the experimental results above.

Manipulations: Based on our preliminary studies we expect that plasmidinjections will be repeated after three weeks for two doses, followed bya single boost with the viral vector three weeks later.

Measures and Outcomes: Sera will be assayed for anti-PC/Kexin IgM, andIgG isotypes by ELISA at 3, 6, and 9 weeks. Isotypes will be determinedby using appropriate anti-mouse IgG isotypes: IgG1, IgG2a, IgG2b, orIgG3 (Pierce, Rockford, Ill.). At 9 weeks, a subgroup will be boostedwith an IN injection of 10⁷ particles of AdKexin (a recombinantadenovirus expressing Kexin). A control group will be left un-boosted,or boosted with AdCD40L. Three weeks after the boost, mice will besacrificed and we will measure anti-PC/Kexin antibodies in serum and BALfluid. Splenocytes and mediastinal lymph node cells will be harvested todetermine the precursor frequency of antigen-specific B-cells byElipsot. Serum antibodies will also be tested for complement-dependentkilling, as well as opsonic phagocytosis and killing of PC in vitro asdescribed above.

Expected Results and Interpretations: Based on our preliminary studiesshowing that AdCD40L delivery to the lungs resulted in a significantincrease in anti-PC/Kexin mucosal IgA and IgG, we expect that CD40L willmost likely be optimal when included in both the prime and the boost. Wealso expect to observe an increase in opsonic killing of PC.

Alternative Approaches: If CD40L co-transduction in vivo is lesseffective than we expect, then we will perform co-culture experiments ofirradiated AdCD40L (or control) modified DCs, pulsed with OVA to induceB cell proliferation and class switching in CD19+ B cells from CD40L KOmice. Particularly we will investigate the ratio of DC's to B cells toinduce differentiation and generation of anti-OVA IgG in vitro. Ifhigher ratios of DCs are required to induce B cell proliferation inCD40L KO mice as compared to control C57BL/6 mice, then we willinvestigate the effects of higher doses of DNA priming, boosting, orboth in vivo. Secondly, if we observe a significant amount of classswitched antibody against PC in CD40L KO mice, we will repeat theexperiment with a PC challenge as outlined above, to examine theefficacy and duration of protection in these mice. If we observeefficacy with the MVA platform, we will also repeat this experimentsubstituting MVA vectors for Ad vectors to examine their efficacy andduration of protection.

Example 24

Examine the role of IL-12p35, IL-12p40, and IL-23 in mucosal primeboosting with recombinant adenovirus virus-based vectors against PC.

Hypothesis: We hypothesize that the co-administration of CD40L withantigen allows for CD4IND B-cell responses in vivo, and that mucosalboosting will enhance mucosal antigen specific B-cell responses andenhance protection against PCP.

Rationale: Our preliminary studies demonstrated that CD40L-modified DCsrequired IL-23 for a functional B-cell recall response to PC antigenwhen the DCs were pulsed with PC organisms. However, these preliminaryresults do not in themselves show whether IL-23 is also required for theprime boost regimen in vivo. If IL-23 is indeed required in vivo, thenIL-23 could be used as a mucosal adjuvant. Secondly, up to 3-7% of thepopulation is heterozygous for a non-synonymous SNP in the IL-23R codingregion. It is possible that this polymorphism could reduce theimmunogenicity of the novel vaccine platform in heterozygousindividuals, or in individuals homozygous for the less frequent allele.

Experimental Groups. Male 6-8 week old BALB/c mice will be CD4-depletedby the administration of 0.3 mg GK1.5 IP by weekly injection. 48 hourslater, mice will be randomized to be vaccinated by the IM injection ofthe two most efficacious Kexin constructs as identified above:pmini-Kexin WT, psec-m in i-Kexin-WT, pm in i-Kexin CO, orpsec-mini-Kexin-CO with CD40L. Control mice will be injected withpBudCD40L with no PC antigen. After 6 weeks the mice will be furtherrandomized to no boost or mucosal boosting with 10⁷ adenovirus encodingthe same antigen construct as the prime, in each case with or without anequal dose of AdCD40L or 10⁷ offowl pox vectors encoding the sameantigen (with our without CD40L). Either an adenovirus vector or an MVAvector will be used, based on their relative efficacy as determined inthe experiments described above. At 9 weeks 50% of the mice will besacrificed to examine serum and mucosal anti-Kexin/PC antibodies as wellas antigen-specific B-cells. The other 50% will be challenged with 2×10⁵PC cysts intratracheally and sacrificed 7 days later to assess the roleof IL-23 in regulating antigen specific B-cell recall responses. Samplesizes will consist of 10 mice per group to give the statisticalresolution to detect a 30% difference in antigen specific B-cellresponses.

Measures and Outcomes: At the time of sacrifice (either week 9 or week10 in PC challenged mice) lungs will be lavaged for anti-PC andanti-Kexin IgA levels. Kexin-specific, IgG-expressing B cells in thespleen and mediastinal lymph nodes will be assayed by Elispot. One lungwill be placed in TRIzol™ reagent for measuring PC mtLSU copy number byreal time PCR. Serum antibodies will be tested for complement-dependentkilling, as well as opsonic phagocytosis and killing of PC in vitro asdescribed above.

Expected Results, Interpretations and Alternative Approaches: We expectto observe the induction of anti-PC and anti-Kexin primary antibodyresponses in the serum of all mice. However, we expect to observe adefect in mucosal boost responses as well as PC recall response in thelungs of IL-23- and IL-12p40-deficient mice. We also expect to observean increase in opsonic killing of PC in IL-23 intact mice (only). Suchresults would be consistent with the expected role of IL-23 in expandingthe B-cell memory pool. If this is indeed the case, then we will repeatthe experiment and examine the effect of adding Ad IL-23 (or MVA IL-23)in the boost along with AdCD40L to determine whether exogenous IL-23 canrestore B-cell expansion in IL-12p40−/− or IL-23p19−/− mice. We havepreviously prepared the AdIL-23 vector. Preliminary studies suggest thatthe AdIL-23 vector will restore B-cell memory responses, at least in thecontext of ex vivo, pulsed DC-based vaccination with IL-23p19−/− DCs.

Example 25

Testing in vivo, CD4IND, pathogen-specific immune responses againstPneumocystis kexin are generated in an SIV model of immunodeficiency inmacaques. We will confirm that the mini-kexin constructs producevaccine-induced immune responses in SIV-infected, CD4-deficientmacaques. Control or SIV-infected macaques will undergo DNA primingfollowed by mucosal boosting 4 weeks after mock or live SIV infection.Outcome measures will include humoral responses to the vaccine and theprevention of Pneumocystis colonization as determined by PCR in BALfluid. Preliminary studies suggest that Pneumocystis colonization occursin up to 80% of SIV-infected (untreated) macaques, compared to 0% innon-SIV infected monkeys.

Example 26

Evaluate the effect of CD40L on monkey DCs.

Hypothesis: We hypothesize that AdhCD40L-transduced (or MVA hCD40Ltransduced) monkey DC's will demonstrate maturation by an increase inClass II MHC expression, and that they will demonstrate activation byenhanced elaboration of IL-12 and IL-23 in culture.

Rationale: Preliminary studies have demonstrated similar results in themouse model. We therefore expect similar results in the macaques.

Experimental Groups. Monkey DC's from juvenile macaques with normal CD4counts will be purified by CD11c+ beads (Mitenyi Biotech) fromperipheral blood. Monkey DC's will be grown in RPMI 1640 and then mocktransfected with PBS; or transfected with AdEGFP or with AdhCD40L atMOIs of 5, 10, 50, and 100. We will carry out similar experiments withMVA CD40L. Sample sizes will consist of 4-6 control macaques; andtransductions will be carried out in triplicate.

Manipulations: CD11c+ DCs will be cultured in RPMI 1640 growth mediumsupplemented with 1% monkey plasma. 50,000 to 100,000 cells will beanalyzed by four-color FACS for each of the following DC markers:anti-HLA-DR, CD80, CD86, and CD25 from B-D Pharmingen, and CD83 fromCoulter. Immature DC's are typically HLA-DR⁺⁺, CD86⁺⁺, CD80^(+/low),CD83^(−/weak), and CD25-, whereas mature DC's are HLA-DR⁺⁺, CD86⁺⁺⁺,CD25⁺⁺, CD80⁺⁺, and CD83⁺⁺. The remaining DC's will be transduced withMOls of 0, 5, 10, 50, or 100 of AdEGFP or AdhCD40L (or MVA vectors) andcultured for 24 hours. The supernatant will be harvested fordeterminations of monkey IL-12 (p40 and p70) and TNF-alpha (Biosource,Camarillo, Calif.). The cells will be stained for maturation markers asoutlined above, as well as for CD40L with clone TRAP-1-PE fromImmunotech (Westbrook ME). IL-23 will be measured by ELISA (BenderMedSystems).

Expected Results and Interpretations: We expect to observedose-dependent transduction of Monkey DC's by both AdEGFP and AdhCD40L,as measured by an increase in the mean channel fluorescence of GFP inAdEGFP-transduced cells, and by an increase in CD40L as measured byTRAP-1 PE staining. Bioactivity of AdhCD40L will be assessed by theability of the vector to selectively induce IL-12, IL-23, and TNF-alphain supernatants from AdhCD40L-transduced DCs. We also expect to observethe maturation of DC's transduced with AdhCD40L as measured by anincrease in mean channel fluorescence in HLA-DR, CD86, and CD83expression. There may also be some increase in HLA-DR, CD86, and CD83expression in Ad EGFP-transduced cells.

Alternative Approaches: We expect efficient transduction of DCs with anMOI of 100. Moreover, human CD40L (h CD40L) has 99% homology with CD40Lfrom monkey. Preliminary studies show that hCD40L induces IL-12p70 inmonkey DCs. Thus we expect that hCD40L should have bioactivity in thissystem. If, however, we observe a defect in DC activation, we willassess hCD40L at the protein level by FASC and at the transcript levelby RT-PCR to verify its expression in DCs with the Ad vector or the MVAvector.

Example 27

Generating Kex1 antigen-specific IgG in SIV-infected macaques byheterologous, prime-boost immunizations.

Hypothesis: We hypothesize that DNA priming followed by heterologousadenovirus or MVA virus boosting will elicit potent anti-Kex1 systemicand mucosal antibody responses and protection against PC colonization.

Rationale: The results of the prior studies will help us choose whichKexin DNA construct to use in macaques, and whether to use adenovirus orMVA as the boost.

Experimental Groups. There will be five vaccine groups:

-   -   1. SIV-uninfected, pneumocystis vaccine with CD40L (n=8)    -   2. SIV-infected, pneumocystis vaccine with CD40L (n=8)    -   3. SIV-uninfected, pneumocystis vaccine without CD40L (n=4)    -   4. SIV-infected, sham vaccine, without PC challenge (n=3)    -   5. SIV-uninfected, sham vaccine, without PC challenge (n=3)        Due to expense and the likelihood that the pneumocystis vaccine        alone (without CD40L) will not elicit strong humoral immunity,        the SIV-uninfected, pneumocystis vaccine without CD40L group        (Group 3) may be omitted from the study.        Groups 4 and 5 will be used as shared controls.

Manipulations, Measurements, and Outcomes: Animals (male juvenile Rhesusmacaques) will be pre-screened by ELISA for anti-PC Kexin titers andanti-adenovirus titers (the latter, if we choose an adenovirusplatform). Only animals with negligible titers, defined as less than1:64 (OD450 cutoff of 0.1), will be enrolled. At −4 weeks monkeys willbe infected with SIVmac251, or mock infected with saline injection. Thedose of virus will be 50 TCID50. Inoculations will be madeintravenously, via the saphenous vein. At week 0, the macaques willundergo a baseline bronchoscopy, they will be assessed for serum and BALanti-Kex 1 antibodies, and they will be assessed for PC colonization bynested and real time PCR. BAL fluid will be obtained by bronchoscopy inanesthetized animals. Plasmid DNA will be administered at a dose of 2 mgIM (2 sites [quadriceps], at 1 mg each in 0.5 ml saline). The plasmidDNA will encode the Kex1 construct that has shown the greatest efficacyin mice, with or without CD40L, as appropriate for each of the groupassignments listed above. DNA will be administered at weeks 0 and 3(i.e., 4 and 7 weeks after mock or live SIV infection). At week 6monkeys will undergo another bronchoscopy, and blood samples will betaken to determine pre-boost anti-Kex1 antibodies in serum and BAL.Following the bronchoscopy, a boost immunization will be administeredintranasally with adenovirus (10¹⁰ pfu) or MVA (10⁸) encoding Kex1 andhCDC40L, or encoding Kex1 alone for monkeys in the no-CD40L group. Athird bronchoscopy will be performed at week 8 to assess the effect ofthe boost on anti-Kex1 IgG in serum, and anti-Kex1 IgA in BAL. Two morebronchoscopies will be performed at weeks 20 and 32 to assess longevityof the antibody responses, as well as the level of Pneumocystiscolonization as determined by both nested and real time PCR. At week 32we will harvest mediastinal lymph nodes for B-cell Elispot assays, andlung tissue for histology. Real-time PCR will also be used to assess PCcolonization in the lungs. To monitor the safety of the approach in themacaque model we will also obtain complete blood counts and serumchemistries, including liver transaminases, at weeks 6, 8, 20, and 32.

Expected Results and Interpretations: We expect that the Kex1/CD40Lvaccination procedure will be safe and will not have associatedhematological or liver toxicity. We expect to observe significantincreases in antigen-specific IgG in both SIV+ and SIV− animalsreceiving hCD40L with Kexl . Moreover, we expect higher anti-Kex1 IgGand IgA levels in SIV− animals receiving the Kexl/CD40L combination ascompared to Kex1 alone. Moreover we expect the addition of a mucosalboost will significantly augment the levels of anti-Kex1 IgG and IgA inBAL fluid (particularly in the Kexl/CD40L groups). We expect to observethat the Kexl/CD40L vaccine will be associated with reduced PCcolonization in the BAL at week 20, and in the BAL and lung tissue atweek 32 in SIV-infected monkeys as compared to the group 4 or 5 animals.

Alternative Approaches: If we observe significant increases in anti-Kex1IgG in SIV− animals but not in SIV+ animals, such an outcome could bedue to inadequate DC homing or an inadequate dose of the boost. If thatshould be the case, then we will repeat a mucosal boost with one loghigher virus dose. Our primary measurement in these experiments will bethe level of PC colonization, as determined by nested and real time PCR.In humans these two assays have over a 93% concordance rate. Ourexpected figure of 80% spontaneous PC colonization in SIV-infectedmacaques is based on observations of animals housed in Pittsburgh, andwe are not yet sure whether a similar level of infection will be seen inmacaques housed at the Tulane Primate Center. To assess, 25 SIV-positiveand 25 SIV-negative lung tissue samples and paired BAL samples will betested to determine the prevalence of PC colonization at necropsy inthese monkeys. Assuming the figure is at least 60% for the SIV-positivecohort, then this set of experiments should provide a meaningfuloutcome. If the figure should be lower, then we will identify lungsamples with high burdens of CP organisms, and isolate organisms fromthose samples to use for inoculation at week 20. Lungs from identifiedanimals will be disrupted and processed as has been previously describedfor murine lung; cysts will be purified by sucrose gradientpurification.

Example 28

Pneumocystis is also a common infection following medically-inducedimmunosuppression, for example in cancer chemotherapy, or in suppressinghost rejection of transplanted tissues or organs. Vaccination withmini-kexin can protect against PCP in such instances.

As a demonstration in a mouse model, we performed DNA plasmid mini-Kexinprime-boost vaccination in wild type mice, and then depleted CD4+T-cells. Depletion of CD4+ T-cells makes unvaccinated mice susceptibleto PCP. Female 6- to 8-week-old C57BL/6 and BALB/c wild type (wt) micewere immunized intramuscularly with a mini Kexin-encoded, pBUD plasmidvector twice, two-weeks apart. The four mini Kexin vectors encodedsecreted (s) or non-secreted versions of mini kexin, eithercodon-optimized (co) or wild type (wt). Two weeks after the second plasmid DNA prime vaccination, the mice were intranasally boosted withrecombinant adenovirus encoding the same type of mini-Kexin as had beenused in the prime vaccination. Because the vaccinated mice initially hadnormal levels of CD4+ T-cells, we did not include CD40L in either thepriming or boosting vector. To artificially induce an immunosuppressedstate, the mice were then depleted of CD4+ T cells by administration ofmonoclonal antibody GK1.5, repeated weekly, starting two weeks after theboost. After one week of CD4+ depletion, the mice were challengedintratracheally with 2×10⁵ PC organisms per mouse. Four weeks later themice were euthanized, and lung tissues were collected for PC organismburden (assayed by real-time PCR).

As shown in FIG. 9, all four miniKexin plasmid vaccines providedsignificant protection against PC infection, as compared to Rag2/gammachain double-knockout mice, which lack B, T, and NK cells (n=5-6 pergroup, * denotes P<0.05, Student's-t-test as compared to RAG2/gamma CKO).

Example 29

Following successful completion of animal trials, vaccines in accordancewith the present invention are tested in human patients in clinicaltrials conducted in compliance with applicable laws and regulations.

Example 30

Detailed Methodology. Except as otherwise stated, the followingmaterials and procedures have been used or will be used in theexperiments described above:

1. Animals. Virus-free BALB/c mice, aged 6-8 weeks, will be purchasedfrom NCI/Charles River. Preliminary experiments have shown that animalsfrom this supplier are not chronically infected with P. carinii.IL-12p35, IL-12p40, and IL-23p19 mice on a C57BL/6 background aremaintained in our laboratory. Homozygous C.B17 scid/scid (scid) micewill be purchased from NCl/Charles River or Taconic Laboratories inGermantown, N.Y. All animals will be housed in separate rooms at the LSUMedical Center Animal Care Facility in HEPA-filtered ventilated racks.Mice are fed autoclaved chow and water ad libitum, and are held in thefacility for least 2 days before initiating treatment. Changes of animalcages, bedding, water bottles, and food will be performed in a laminarflow hood. Access to the room is limited to specific laboratorypersonnel and animal care personnel; gown and gloves are required forall workers entering the room. It is estimated that we will use a totalof 1368 mice in the experiments described. Macaques are housed at theTulane National Primate Research Center. Where applicable, monkeys arepre-anesthetized with acepromazine (0.2 mg/kg, i.m.) and sedated withketamine-HCl (10 mg/kg, i.m.) for bronchoscopy and blood sampling.Bronchoscopy will be performed with topical anesthesia with 2%xylocaine.

2. Monitoring Animal Health. Sentinel DBA mice are co-housed in the sameroom as the experimental mice, with bedding regularly taken from thecages of P. carinii-infected and scid mice. The sentinel mice aresacrificed quarterly and tested for antibody titers to a variety ofmurine viruses and pathogens. Regular consultation with veterinary staffis used to assure and to confirm specific-pathogen-free conditions forthe experimental animals.

3. Maintenance of P. carinii in scid or CD40L knockout mice. To assure aconsistent supply of P. carinii, the P. carinii organisms will bepassaged through the lungs of C.B17 scid/scid (on a BALB/c background)or CD40L KO mice (on a C57BL/6 background). We presently maintain abreeding colony of PC-free and PC-infected CD40L KO mice in separaterooms. Groups of scid or CD40L KO mice will be inoculated with P.carinii organisms as described below. Inoculated mice will be housed inventilated racks for 4-6 weeks before being sacrificed to harvest P.carinii from lung tissue.

4. Inoculation of Mice with P. carinii organisms. P. carinii organismsused for inoculation will be prepared from homogenized lungs ofchronically infected scid or CD40L KO mice. We have previously usedathymic mice, but have found that scid mice develop more consistent andintense infections. The C.B17 scid mouse strain is allotype co-isogenicto BALB/c mice. Briefly, lung tissue will be obtained from scid or CD40LKO mice chronically infected with P. carinii. The lungs will be frozenfor 30 minutes, and then disrupted mechanically in a Stomacher™ 80Biomaster. The disrupted lung tissue will be filtered through gauze andadjusted to a level of 2×10⁶ cysts/ml, as assessed by DiffQuik™Romanowski staining. P. carinii will then be injected into the tracheaof lightly anesthetized BALB/c mice by passing a blunt needle into thetrachea per os and then threading a catheter through the needle into thelow trachea. Each mouse will receive 0.1 ml of inoculum (2×10⁵ cysts),followed by 0.8 ml of air. To assure viability of the organisms, theinoculum will be injected into recipient mice on the day of preparation.Lung homogenates containing P. carinii will be routinely checked forendotoxin contamination using the Whitaker endotoxin assay. The inoculumwill also be quantified using a TaqMan™-based assay for rRNA copynumber. For antigen preparation, PC organisms will isolated from lungtissue of C.B-17 scid mice (for experiments in BALB/c mice) or CD40L KOmice (for experiments in C57BL/6 mice) that were previously inoculatedwith PC. PC organisms will be purified by differential centrifugation,and protein antigen will be produced by sonication for 5 minutes.

5. Examination of Lung Tissue for P. carinii Infection. Lung tissue willbe fixed in formalin and stained with Gomori's methenamine silver andhematoxyl in/eosin.

6. Controls for Bacterial/FungalNiral Infection. Random samples of lungtissue from control and experimental mice will be cultured to excludethe possibility of intercurrent bacterial or fungal infection. Inaddition, when the experimental design permits, touch preps will be madeof lung tissue prior to formalin fixation and gram staining to look forbacterial infection. Also, co-housed sentinel DBA mice are routinelymonitored for serologic titers against common viral pathogens, includingSendai and Mouse Hepatitis Virus.

7. Depletion of host CD4+ lymphocytes. The hybridoma GK1.5 (rat antiCD4)was originally obtained from the American Type Culture Collection(Manassas, Va.) and is maintained in the LSU Monoclonal AntibodyFacility. GK1.5 is a rat IgG2b monoclonal antibody. Antibodies from thishybridoma are prepared from ascites in athymic mice. The antibodies arepartially purified by ammonium sulfate precipitation, dialyzed againstphosphate buffered saline, and quantified by protein electrophoresis andoptical density. To deplete mice of CD4+ T lymphocytes, mice willreceive an intraperitoneal injection of 0.3 mg anti-CD4 monoclonalantibody in 0.2 ml PBS. Control mice will receive an equal volume of ratIgG. Depletion of CD4+ lymphocytes will be checked by flow cytometricanalysis of splenocytes or peripheral blood as described below.Depletion of the appropriate lymphocyte subset will be maintained byweekly administration of the antibodies for the course of theexperiment.

8. RNA isolation and TaqMan™ probes and primers for PC rRNA. Total RNAis isolated from the right lung of infected mice by a single step methodusing TRIzol™ reagent (Life Technologies, CA, USA). As a standard forthe assay, a portion of PC muris rRNA (GenBank Accession No. AF257179)is cloned into PCR2.1 (Invitrogen, Carlsbad, Calif.), and PC rRNA isproduced by in vitro transcription using T7 RNA polymerase. The templateis digested with RNase-free DNase, quantitated by spectrophotometry andaliquoted at −80° C. until used. The TaqMan™ PCR primers for mouse PCrRNA are 5′-ATG AGG TGA AAA GTC GAA AGG G-3′ (SEQ ID NO. 7) and 5′-TGATTG TCT CAG ATG AAA AAC CTC TT-3′ (SEQ ID NO. 8) . The probe is labeledwith a fluorescent reporter dye, 6-carboxyfluorescein (FAM), and thesequence is 6FAM -ACAGCCCA GAATAAT GAATAAA GTTCCTCAATT GTTAC-TAM RA (SEQID NO. 9). (TAMRA=tetramethyl-6-Carboxyrhodamine.) Real-time PCR iscarried out using one-step TaqMan™ RTPCR reagents (Applied Biosystems,Foster City, Calif.). The PCR amplification is performed for 40 cycles:94° C. for 20 s and 60° C. for 1 min, in triplicate using the ABI Prism7700 SDS. The threshold cycle CT values are averaged from the valuesobtained from each reaction, and data are converted to rRNA copy numberusing a standard curve. This assay has a correlation coefficient greaterthan 0.98 over 8-logs of PC RNA concentration, and is known to correlatewith viable PC since either heat killing or exposure toTrimethoprim/Sulfamethoxazole ablates the signal.

9. Pneumocystis viability assay. Macrophages (10⁶/ml) suspended in avolume of 100 μl of RPMI 1640 medium containing FCS are co-cultured inround-bottom 96-well plates with PC (2×10⁴ cysts/ml, 50 μl), yielding aneffector-to-total-PC organism ratio of 1:1 (estimated 1:10 cyst totrophozoite ratio). Before addition of PC, organisms will bepreopsonized with 50 μl of serially diluted serum or normalized BAL, or50 μl of DMEM plus 10% FCS. Included as a viability control are PCorganisms incubated with control medium, DMEM plus 10% FCS. The platesare spun at 2500 rpm to pellet the PC organisms. The supernatants andcell pellets are collected, and total RNA is isolated using TRIzol™ LSreagent (Invitrogen Life Technologies). Viability of the PC is analyzedwith real-time PCR measurement of PC large subunit rRNA copy number(GenBank accession number AF257179), and quantified against a standardcurve. This method detects viable PC organisms, as evidenced by loss ofdetectable PC rRNA in heat-killed organisms or those exposed totrimethoprim/sulfamethoxazole.

10. PC Kex 1 ELISA. To determine anti-PC or Kex1 IgG titers, ELISAplates (Corning, N.Y.) are coated with 100 ng of PC antigen or Kex 1antigen (provided by Dr. Karen Norris, University of Pittsburgh) perwell in carbonate buffer at pH 9.5, and held overnight. Plates arewashed with PBS+0.05% Tween-20 (wash buffer) and blocked with bovineserum albumin and 2% milk. After washing, serial dilutions of serum willbe added to each well and incubated for one hour at room temperature.Then, after washing, 100 μl of 1:1000 alkaline phosphatase conjugatedgoat anti-mouse IgG or IgA (Bio-RAD, Hercules, Calif.) will be added andincubated for one hour at room temperature. Then, after washing, theplates are developed using Sigma 104 substrate tablets in diethanolaminebuffer, and absorbance is measured at 490 nm. Anti-PC and anti-Kex1specific mouse IgG isotypes will be assayed. For macaque antibodies weuse anti-Rhesus IgA and IgG.

11. Bronchoalveolar lavage. Lavaged lymphocytes will be obtained bybronchoalveolar lavage of mice anesthetized with intraperitonealpentobarbital. This technique has been previously used in our laboratoryto recover lung cells from mice, rats, and monkeys. For mouse studies,the lungs will be lavaged through an intratracheal catheter with warm(37° C.) calcium- and magnesium-deficient PBS supplemented with 0.5 mMEDTA. A total of 11 ml will be used for each mouse in 0.5 ml increments,with a 30 second dwell time. This technique recovers 0.5-1×10⁶ cellsfrom normal animals, of which greater than 95% are alveolar macrophages,with greater than 95% viability as measured by trypan blue exclusion. Inmice inoculated with P. carinii, total cell count can be as high as4-6×10⁶, and the percentage of lymphocytes contained within the lavagedcells is as high as 50%. For some studies, the first 1.0 ml of BAL fluidmay be frozen for cytokine analysis, or BAL fluid may be concentrated torecover detectable cytokine or IgA.

12. Retrieval of hilar and paratracheal lymph nodes. Hilar lymph nodesand paratracheal (mediastinal) lymph nodes will be resected understerile conditions from mice given a lethal dose of pentobarbital. Thismethod has been used to study draining lymph node cells from micechallenged with antigen. The lymph nodes will be passed through asterile mesh screen into culture medium, and adjusted for cell numberwith a hemacytometer. Using this technique, approximately 12-15×10⁶cells are recovered from a mouse inoculated with P. carinii. More than90% of these cells are lymphocytes as measured by Diff-Quik™ staining.Cells will be processed for flow cytometry as outlined above.

13. B-cell Elipsots. To determine precursor frequency of Kex1 specificIgG B-cells, we will perform ELISPOT assays using FACS-sorted B cellpopulations. 96-well PVDF filter plates will be coated with Kex1, andserial dilutions of sorted B cells will be applied to the wells.Anti-Kex1 antibodies captured on the filter will be visualized bystaining with an AP-conjugated anti-IgG or IgA secondary antibody. Afterdeveloping with chromogenic substrate, the plates will be counted usingan automated plate reader, and the percentage of antibody producingcells in each subset will be calculated.

14. DNA Vaccination. For intramuscular delivery, mice are anesthetizedwith isoflurane. Then 100 microgram of the DNA vaccine is delivered in100 μl of normal saline to the tibialis muscle (i.e., 50 μl per hindleg) using a fine-needle (30 G) tuberculin syringe. If needed, we willfollow immediately by mild electroporation using a BTX ECM 830electroporator apparatus with caliper electrodes (Harvard Biosciences).Immediately following injection of DNA to each leg, the calipers will beset to 4-5 mm and placed tightly on either side of the tibialis. Themachine will be discharged twice, resulting in 2×20 millisecond pulsesof 150V at an interval of 1 sec.

15. Statistical Analysis. Data will be analyzed using StatViewstatistical software (Brainpower Inc., Calabasas, Calif.). Comparisonsbetween groups will be made with the Student's t-test, and comparisonsamong multiple groups will be made with analyses of variance andappropriate follow-up testing. The Mann-Whitney test or the Wilcoxsonpaired sample test will make ordinal comparisons. Significance will betaken as p<0.05.

Example 31 Use and Care of Vertebrate Animals

1. Justification for the Use of Experimental Animals: There are noalternatives to the use of live animals to study host defensemechanisms, nor to study vaccine responses against Pneumocystis.Pneumocystis cannot be reliably maintained in vitro, so research withthis pathogen requires the use of animal models of infection. Rhesusmonkeys are being used because of the similarities between infection ofthis species with simian immunodeficiency virus (SIV) and humaninfection with HIV/AIDS.

2. Veterinary Care of Experimental Animals: Mice will be housed in aseparate room at the LSU Medical Center Animal Care Facility inventilated rack caging. This facility is State-licensed and fullyaccredited by the American Association for Accreditation of LaboratoryAnimal Care (AAALAC). Animals are housed under specific-pathogen-freeconditions. Personnel access to the animal room is limited. All food,cages, water, and bedding is autoclaved prior to use. Gowns, gloves, andmask are required to handle the animals. All cage, food, and beddingchanges take place in a laminar flow biosafety hood in the same room.Sentinel animals are housed in the same room with sample bedding fromthe immunosuppressed animals. Serum from these sentinel animals isroutinely screened for a battery of murine pathogens. A veterinarianoversees the facility.

Veterinary care of the macaques at the Tulane National Primate ResearchCenter will be handled similarly. The animals undergoing study will bemonitored closely for food and water intake, and routine blood work willbe limited to 40 cc/month. The primate center has strict protocols inplace to euthanize SIV-infected animals when weight loss or otherclinical parameters indicate significant morbidity. Animals to beinfected with SIV will receive 50 TCID50 of strain SIVmac251, apathogenic strain to which animals have had a median survival of 210days in past studies. Basic monitoring will include: 1) twice dailyobservations by a trained animal care technician, 2) physicalexaminations including blood sampling prior to SIV inoculation, afterSIV inoculation, and at bi-monthly intervals thereafter. All physicalexaminations and invasive procedures will be performed on anesthetizedanimals. The anesthetic used will either be a combination ofketamine-HCl (10 mg/kg, i.m.) and acepromazine (0.2 mg/kg, i.m.), orTelazol (5 mg/kg of Tiletamine and Zolazepam).

Containment practices at the Tulane Primate Center are in accordancewith the recommended guidelines in the Center for Disease ControlMorbidity and Mortality Weekly Report, vol. 31 (#43) “AIDS: Precautionsfor Clinical and Laboratory Staff, pp. 577-580 (1982); vol. 32 (#34)“AIDS: Precautions for Health Care Workers and Applied Professionals”pp. 577-580 (1983); and the Biosafety in Microbiological and BiomedicalLaboratories Guidelines, First edition (1984).

3. Experimental Procedures involving Live Animals:

a. Intratracheal Inoculation with P. carinii Organisms: Pneumocystiscarinii organisms will be obtained from the lungs of chronicallyinfected scid or CD40L KO mice. Organisms for this colony of infectedmice were originally obtained from the Fox Chase Cancer Center inPhiladelphia, Pa. Mice will be sacrificed by a lethal (400 mg/kg) doseof IP pentobarbital, followed by exsanguination once they are deeplyasleep. The lungs will then be removed aseptically, and Pneumocystiscarinii organisms will be recovered for injection into other scid mice(to maintain the organisms) or into BALB/c mice (to conduct the proposedexperiments). The Pneumocystis carinii organisms will be injected in avolume of 0.1 ml into the tracheas of mice lightly anesthetized withinhaled isoflurane. Once the animals are asleep, the animals are brieflysuspended by their teeth, the tongue is gently pulled forward withtweezers, and the inoculum is injected into the lungs using a blunt 18 gneedle. These inoculations do not appear to cause undue discomfort orpain, and are (themselves) associated with minimal mortality. Once theinjected animals have recovered from the anesthesia, they (initially)appear healthy.

Mice will not receive analgesics after intratracheal inoculations forthe following reasons: a) Many analgesics are known to alter the hostresponse to infection and endotoxin. b) There is no evidence that miceundergoing this procedure experience pain or discomfort. c) Mostanalgesics have an extremely short half life in rodents, which wouldnecessitate multiple injections, that could become a stress inthemselves.

b. Depletion of CD4+ lymphocytes: Mice will be depleted of lymphocytesby weekly intraperitoneal injections (0.2 ml) with an anti-CD4monoclonal antibody. This procedure effectively depletes treated animalsof targeted T lymphocytes in blood and lymphoid tissue with minimalmorbidity and no mortality (in itself). Treated animals do not loseweight and they (initially) appear healthy.

c. DNA vaccination and mucosal boosting: Mice will be injected underisoflurane anesthesia with endotoxin-free plasmid DNA, 100 μg, split intwo injections, one into each tibialis anterior muscle. Mucosal boostingis performed by the intranasal administration of virus under isofluraneanesthesia.

Example 32 Uses Against Other Pathogenic Fungi

The methods and constructs of this invention are also expected to beeffective in conferring immunity against at least some other pathogenicfungi, for example Candida glabrata and Candida albicans, both of whichare human pathogens. A BLAST comparison of the kexin amino acidsequences in these two species versus that of P. carinii showed 53%homology with that of C. glabrata and 50% with that of C. albicans.Effectiveness against other fungal species with ˜40% or more amino acidsequence homology is expected.

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63. Duerr, R. H., Taylor, K. D., Brant, S. R., Rioux, J. D., Silverberg,M. S., Daly, M. J., Steinhart, A. H., Abraham, C., Regueiro, M.,Griffiths, A. et al 2006. A Genome-Wide Association Study IdentifiesIL23R as an Inflammatory Bowel Disease Gene. Sci 314:1461-1463.

64. Happel, K. I., Lockhart, E. A., Mason, C. M., Porretta, E.,Keoshkerian, E., Odden, A. R., Nelson, S., and Ramsay, A. J. 2005.Pulmonary interleukin-23 gene delivery increases local T-cell immunityand controls growth of Mycobacterium tuberculosis in the lungs. InfectImmun 73:5782-5788.

65. Reay, J., Kim, S. H., Lockhart, E., Kolls, J., and Robbins, P. D.2009. Adenoviral-mediated, intratumor gene transfer of interleukin 23induces a therapeutic antitumor response. Cancer Gene Ther.

66. Morelli, A. E., Larregina, A. T., Ganster, R. W., Zahorchak, A. F.,Plowey, J. M., Takayama, T., Logar, A. J., Robbins, P. D., Falo, L. D.,and Thomson, A. W. 2000. Recombinant adenovirus induces maturation ofdendritic cells via an NF- kappaB-dependent pathway. J Virol.74:9617-9628.

67. Kikuchi, T., Moore, M. A., and Crystal, R. G. 2000. Dendritic cellsmodified to express CD40 ligand elicit therapeutic immunity againstpreexisting murine tumors. Blood 96:91-99.

68. Zhong, L., Granelli-Piperno, A., Pope, M., Ignatius, R., Lewis, M.G., Frankel, S. S., and Steinman, R. M. 2000. Presentation of SIVgag tomonkey T cells using dendritic cells transfected with a recombinantadenovirus. Eur. J Immunol. 30:3281-3290.

69. Neeson, P., Boyer, J., Kumar, S., Lewis, M. G., Mattias, L., Veazey,R., Weiner, D., and Paterson, Y. 2006. A DNA prime-oral Listeria boostvaccine in rhesus macaques induces a SIV-specific CD8 T cell mucosalresponse characterized by high levels of alpha4beta7 integrin and aneffector memory phenotype. Virology 354:299-315.

70. Shean, M. K., Baskin, G., Sullivan, D., Schurr, J., Cavender, D. E.,Shellito, J. E., Schwarzenberger, P. O., and Kolls, J. K. 2000.Immunomodulation and adenoviral gene transfer to the lungs of nonhumanprimates. Hum. Gene Ther. 11:1047-1055.

71. Sullivan, D. E., Dash, S., Du, H., Hiramatsu, N., Aydin, F., Kolls,J., Blanchard, J., Baskin, G., and Gerber, M. A. 1997. Liver-DirectedGene Transfer in Non-human Primates. Hum. Gene Ther. 8:1195-1206.

Publications by the Inventors and Their Colleagues

1. Steele C, Marrero L, Shellito J E, Kolls J K. Alveolarmacrophage-mediated killing of Pneumocystis carinii f. sp. murisinvolves pattern recognition by the Dectin-1 beta-glucan receptor. J.Exp Med. 2003; 198:1677-1688

2. Happel K I, Zheng M, Quinton L J, Lockhart E, Ramsay A J, Shellito JE, Schurr J R, Bagby G J, Nelson S, Kolls J K. Cutting Edge: Roles ofToll-Like Receptor 4 and IL-23 in IL-17 Expression in Response toKlebsiella pneumoniae Infection. J. Immunol 2003;170:4432-4436.

3. Kolls J K, Kanaly S T, Ramsay A J. Interleukin 17: an emerging rolein lung Inflammation. Am J Respir Cell Mol Biol 2003 January;28(1):9-11

4. McAllister F, Steele C, Zheng M, Young Erana, Shellito J E, MarreroL, Kolls J K. Tc1 CD8+ T-cells are effector cells against Pneumocystisin mice. J Immunol 2004;172:1132-1138.

5. Kolls J K and Linden A. IL-17 family members and Inflammation.Immunity. 2004 October;21(4):467-76.

6. Steele C, Shellito J E, Kolls J K. Immunity against the opportunisticfungal pathogen Pneumocystis. Medical Mycology 2004;43:1-19.

7. Schurr J R, Young E, Byrne P, Steele C, Happel K, Shellito J E, KollsJ K. Central role of TLR4 signaling and host defense in experimentalgram negative pneumonia. Infection and Immunity 2005;73:532-545.

8. Mc Allister F, Henry A, Kreindler J L, Dubin P J, Ulrich L, Steele C,Finder J D, Pilewski J M, Carreno B, Goldman S J, Pirhonen J, and KollsJ K. Role of IL-17A, IL-17F and the IL-17 receptor in regulatingGro-alpha and G-CSF in Bronchial Epithelium: implications for airwayinflammation in cystic fibrosis. J Immunol 175(1):404-12, 2005.

9. Happel K I, Dubin P J, Zheng M, Ghilardi N, Lockhart C, Quinton L J,Odden A R, Shellito J E, Bagby G J, Nelson S, Kolls J K Divergent rolesof IL-23 and IL-12 in host defense against Klebsiella pneumoniae J ExpMed 2005;202:761-769.

10. Ruan S, Young E, Luce M J, Reiser J, Kolls J K, Shellito J E.Conditional expression of interferon-gamma to enhance host responses topulmonary bacterial infection. Pulmonary Pharmacology and Therapeutics.2005;19:251-257.

11. Zheng M, Ramsay A J, Robichaux M B, Norris K A, Kliment C,Crowe C,Rapaka R R, Steele C, McAllister F, Shellito J E, Marrero L,Schwarzenberger P, Zhong Q, and Kolls J K. CD4+ T cell-independent DNAvaccination against opportunistic infections J. Clin. Invest., 2005;115: 3536-3544

12. McAllister F, Steele C, Zheng M, Shellito J E, Kolls J K. In vitroeffector activity of Pneumocystis-specific T cytotoxic-1 CD8+ T-cells:role of GM-CSF. Infec Immun 2005;73:7450-7457.

13. McAllister F, Ruan S, Kolls J K, Shellito J E. CXCR3 and IP-10Pneumocystis pneumonia. J. Immunology 2006;177:1846-1854.

14. McKinley L, Logar A J, McAllister F, Zheng M, Steele C, and Kolls JK. Regulatory T Cells Dampen Pulmonary Inflammation and Lung Injury inan Animal Model of Pneumocystis Pneumonia. J. Immunol. 177(9):6215-6226,2006.

15. Rapaka R R, Goetzman E S, Zheng M, Vockley J, McKinley L, Kolls J K,Steele C. Enhanced defense against Pneumocystis carinii mediated by anovel dectin-1 receptor Fc fusion protein. J Immunol. 178(6):3702-12,2007.

16. Hsu H C, Yang P, Wang J, Wu Q, Myers R, Chen J, Yi J, Guentert T,Tousson A, Stanus A L, Le T V, Lorenz R G, Xu H, Kolls J K, Carter R H,Chaplin D D, Williams R W, Mountz J D. Interleukin 17-producing T helpercells and interleukin 17 orchestrate autoreactive germinal centerdevelopment in autoimmune BXD2 mice. Nat Immunol. 2008February;9(2):166-75

17. Aujla S, Chan Y C, Zheng M, Fei M, Askew D J, Pociask D A, ReinhartT A, McAllister F, Edeal J, Gaus K, Husain S, Kreindler J L, Dubin P J,Pilewski J M, Myerburg M M, Mason C A, Iwakura Y, and Kolls J K. IL-22mediates mucosal host defense against gram negative bacterial pneumonia.Nat Med. 2008 March;14(3):275-81.

18. Raffatellu M, Santos R L, Verhoeven D, Wilson R P, Winter S E,Godinez I, Sankaran S, Paixao T, George M D, Gordon M A, Kolls J K,Dandekar S, and Baumler A J. IL-17 orchestrates a mucosal responseagainst Salmonella dissemination from the gut. Nat Med. 2008April;14(4):421-8.

19. Ruan S, McKinley L, Zheng M, Rudner X, Kolls J K, Shellito J E.Interleukin-12 and host defense against murine Pneumocystis pneumonia.Infection and Immunity 2008;76: 2130-2137.

20. Ouyang W, Kolls J K, Zheng Y. The biological functions of T helper17 cell effector cytokines in inflammation. Immunity. 2008April;28(4):454-67.

21. Kolls J K, McCray P B Jr, Chan Y R. Cytokine-mediated regulation ofantimicrobial proteins. Nat Rev Immunol. 2008 November;8(11):829-35.

22. Chan Y R, Liu J, Pociask D, Zheng M, Mietzner T A, Berger T, Mak T,Clifton M, Strong R K, Ray P, Kolls J K. Lipocalin 2 is required forpulmonary host defense against Klebsiella infection. J. Immunol. 2009,182:4493-4494.

Miscellaneous

The complete disclosures of all references and publications cited inthis disclosure are hereby incorporated by reference in their entirety,as is the entire disclosure of priority application 61/294,252. In theevent of an otherwise irreconcilable conflict, the present specificationshall control.

1. The protein mini-Kexin (SEQ ID NO 5).
 2. An isolated nucleic acidencoding the protein of claim
 1. 3. The nucleic acid of claim 2, whereinsaid nucleic acid has sequence SEQ ID NO 1 or SEQ ID NO
 2. 4. Thenucleic acid of claim 2, wherein the codons of said nucleic acid areoptimized to enhance expression in mammalian cells.
 5. The nucleic acidof claim 4, wherein said nucleic acid has sequence SEQ ID NO 3 or SEQ IDNO
 4. 6. A fusion protein that comprises mini-Kexin (SEQ ID NO 5), butthat does not comprise the entire Kexin protein; and that furthercomprises a leader that promotes secretion of said fusion protein from amammalian cell.
 7. An isolated nucleic acid encoding the fusion proteinof claim
 6. 8. The fusion protein of claim 6, wherein said leader is anIgGk leader, and wherein the fusion protein has sequence SEQ ID NO
 6. 9.An isolated nucleic acid encoding the fusion protein of claim
 8. 10. Thefusion protein of claim 6, wherein said fusion protein comprisesmini-Kexin and further comprises CD40L.
 11. An isolated nucleic acidencoding the fusion protein of claim
 10. 12. A vaccine comprising theprotein of claim 1, and additionally comprising an adjuvant.
 13. Thevaccine of claim 12, wherein the adjuvant comprises CD40L.
 14. A vaccinecomprising the nucleic acid of claim 2, and additionally comprising anisolated nucleic acid that encodes an adjuvant.
 15. The vaccine of claim14, wherein the encoded adjuvant comprises CD40L.
 16. A vaccine thatcomprises a live virus containing the nucleic acid of claim
 2. 17. Amethod of immunizing a mammalian patient against infection byPneumocystis, said method comprising administering to the patient thevaccine of claim
 12. 18. A method of immunizing a mammalian patientagainst infection by Pneumocystis, said method comprising administeringto the patient the vaccine of claim
 14. 19. A method of immunizing amammalian patient against infection by Pneumocystis, said methodcomprising administering to the patient the fusion protein of claim 10.20. A method of immunizing a mammalian patient against infection byPneumocystis, said method comprising administering to the patient thenucleic acid of claim
 11. 21. The method of claim 20, wherein thepatient is a human.
 22. The method of claim 21, wherein the patient isimmunocompromised.
 23. The method of claim 21, wherein the patient isnot suffering adverse effects of Pneumocystis infection, and whereinsaid method affords protection against future infection by Pneumocystis.24. The method of claim 21, wherein the patient is suffering adverseeffects of Pneumocystis infection, and wherein said method amelioratesthe Pneumocystis infection.
 25. The method of claim 21, wherein thepatient is immunized a plurality of times to provide a stronger immuneresponse than would be provided by a single immunization.
 26. The methodof claim 21, wherein said method comprises one or more primevaccinations and one or more boost vaccinations; and wherein the primeand boost vaccinations differ from one another in the location ofadministration, or the composition of the vaccine, or both.
 27. Themethod of claim 26, wherein the prime vaccination comprisesintramuscular vaccination with a plasmid vaccine; and wherein the boostvaccination comprises intranasal vaccination or other mucosalvaccination with an adenovirus vaccine.
 28. A method of immunizing amammalian patient against fungal infection, said method comprisingadministering to the patient the vaccine of claim
 12. 29. A method ofimmunizing a mammalian patient against fungal infection, said methodcomprising administering to the patient the vaccine of claim 14.